Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells

ABSTRACT

The invention relates to methods for producing papilloma-derived nanosphere particles that contain therapeutic, diagnostic, or other agents. The invention also provides nanosphere particle preparations that are useful for selectively delivering therapeutic, diagnostic, and/or other agents to cancer cells of subjects without eliciting a serotype-specific immunogenic response in the subjects.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/376,408, filed Aug. 1, 2014, which is a national stage filing underU.S.C. §371 of International application number PCT/US2013/025230, filedFeb. 7, 2013, which was published under PCT Article 21(2) in English andclaims the benefit under 35 U.S.C. §119(e) of U.S. provisionalapplication Ser. No. 61/596,042, filed Feb. 7, 2012, each of which isherein incorporated by reference in its entirety.

FIELD OF INVENTION

Embodiments described herein relate to the fields of molecular biologyand medicine.

BACKGROUND OF THE INVENTION

Many therapeutic agents cannot be delivered effectively to treatcancerous cells by conventional means such as ingestion, injection,inhalation, and topical application because many of these agents aresubject to rapid degradation. Further, direct systemic administration ofmany therapeutic agents often causes detrimental side-effects. Forexample, those agents that target actively dividing cells are not ableto discriminate between actively dividing cancer cells and activelydividing healthy cells. Thus, in the process of destroying or inhibitingthe rapidly dividing cancer cells, many of the healthy cells are alsodamaged.

Accordingly, there is an unmet need for targeting of solid tumor cellsfor the treatment of malignant diseases that will show an affinity forcancer cells, deliver therapeutic payloads that inhibit proliferationand/or destroy cancerous tumor cells without inhibiting and/ordestroying normal cells.

SUMMARY OF INVENTION

In some embodiments, aspects of the invention relate to methods andcompositions for delivering therapeutic and/or diagnostic agents to atarget tissue in a subject (e.g., mammal such as human). In someembodiments, methods and compositions are provided for creating andusing virion-derived protein nanosphere particles (NSPs) that exhibitsurprising selectivity for delivering molecules to tumors withouttargeting healthy tissue and without producing a serotype-specificimmunogenic response in the subject. Accordingly, NSPs described hereinare useful for delivering toxic agents to tumors with reduced risk tohealthy cells. However, NSPs described herein also may be used toselectively deliver other therapeutic and/or diagnostic agents asaspects of the invention are not limited in this respect.

In some embodiments, aspects of the invention are based on the selectivetropism of NSPs described herein for tumor cells. The proliferation oftumor cells is characterized by inflammation of tumor sites, the abilityof tumor cells to evolve HSPG (heparan sulphate proteoglycans) in asimilar manner to basal membrane Keratinocytes, and the presence ofvarious growth factor receptors known to congregate at the surface oftumor cells. In some embodiments, nanosphere particles described hereinare attracted to one of more of these tumor specific properties.

Thus, in some aspects, provided herein are methods of selectivelydelivering an agent to at least one tumor (e.g., one or more) in asubject, the method comprising administering a tumor tropic nanosphereparticle to a subject, wherein the tumor tropic nanosphere particlecomprises mutated or modified human papillomavirus (HPV) L1 capsidprotein without a heterologous targeting agent, is associated with anagent, and is free of host cell nucleic acid and viral nucleic acid. Insome embodiments, the nanosphere particle comprises wild-type HPV L2capsid proteins. In some embodiments, the mutated HPV L1 capsid proteinhas an amino acid sequence alteration that modifies in the subject HPVserotype-specific immunogenicity of the capsid protein relative to anaturally-occurring capsid protein. In some embodiments, the mutated HPVL1 capsid protein has an amino acid sequence alteration that prevents inthe subject HPV serotype-specific immunogenicity of the capsid proteinrelative to a naturally-occurring capsid protein. In some embodiments,the mutated HPV L1 capsid protein is a mutated HPV-16 or HPV-31 L1capsid protein. In some embodiments, the mutated HPV L1 capsid proteincomprises the amino acid sequence of SEQ ID NO:2.

In some embodiments, the modified HPV L1 capsid protein has an aminoacid that is PEGylated.

In some embodiments, the at least one tumor is pre-malignant ormalignant. In some embodiments, the at least one tumor is metastatic. Insome embodiments, the at least one tumor is a non-mucosal tumor. In someembodiments, the at least one tumor is a solid tumor. In someembodiments, the at least one tumor contains cancer stem cells. In someembodiments, the at least one tumor is an astrocytoma, an atypicalteratoid rhaboid tumor, a bone or connective tissue tumor, a brain cyst,a choroid plexus tumor, a craniopharyngioma, an ependymoma, a germ celltumor, a glioblastoma, a glioma, a hemangioma, a juvenile pilocyticastrocytoma, a lipoma, a lymphoma, a medulloblastoma, a meningioma, aneurofibroma, a neuronal tumor, a mixed neuronal-glial tumor, anoligoastrocytoma, and oligodendroglioma, a pineal tumor, a pituitarytumor, a primitive neuroectodermal tumor or a schwannoma. In someembodiments, the at least one tumor is located in the breast, cervix,ovary, testis, prostate, lung, lymph node, stomach, intestine, colon,brain, or a combination thereof.

In some embodiments, the agent is a therapeutic agent. In someembodiments, the agent is an anti-cancer agent. In some embodiments, theanti-cancer agent is delivered to the subject in a therapeuticallyeffective amount to treat (e.g., shrink or irradicate) the tumor.

In some embodiments, the therapeutic agent is an inorganic molecule, anorganic molecule or a biologically active molecule. In some embodiments,the therapeutic agent comprises a small molecule, a protein, a peptide,an antibody, a toxin, a nucleic acid, a radioisotope, a radiolabeledmolecule, a metal, an inducer of DNA methylation, a modulator of geneexpression, an immune modulator, an enzyme inhibitor, a kinaseinhibitor, an apoptosis inducer, a metabolism inhibitor, or anycombination thereof. In some embodiments, the therapeutic agent is aradioisotope or a radiolabeled molecule. In some embodiments, thenucleic acid is an siRNA molecule, an shRNA molecule, a microRNA, a longnon coding RNA, a hybrid DNA-RNA, a DNA molecule, an antisense molecule,a viral gene cassette, or any combination thereof.

In some embodiments, the agent is a diagnostic agent. In someembodiments, the diagnostic agent is an imaging agent or a contrastagent. In some embodiments, the diagnostic agent is labeled with adetectable label. In some embodiments, the detectable label is afluorescent or radioactive label.

In some embodiments, the agent is encapsulated with the nanosphereparticle. In some embodiments, the agent is mixed with the capsidproteins in the nanosphere particle. In some embodiments, the agent ischemically linked to an amino acid of one or more capsid proteins in thenanosphere particle.

In some embodiments, the mutated HPV L1 capsid protein is expressed,isolated and purified as a monomer or as an oligomeric capsomere from ahost cell expression system. In some embodiments, the mutated HPV L1capsid protein and the L2 capsid protein are expressed from differentnucleic acids in a host cell expression system. In some embodiments, thehost cell expression system is a bacterial, a yeast, an insect, a plantor a mammalian host cell expression system. In some embodiments, thebacterial host cell expression system is an Escherichia coli host cellexpression system. In some embodiments, the capsid proteins and/orcapsomeres are assembled in vitro to form the nanosphere particle.

In some aspects, provided herein are methods of producing a nanosphereparticle for selectively delivering a therapeutic or diagnostic agent toa tumor in a subject, the method comprising, providing mutated ormodified human papillomavirus (HPV) L1 capsid protein that is free of(without detectable amounts or the detectable level is very low, e.g.,less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or lessthan 0.01% relative to the capsid proteins) host cell nucleic acid andviral nucleic acid, and reassembling the capsid protein in the presenceof a therapeutic agent or diagnostic agent.

In some aspects, provided herein are methods of producing a nanosphereparticle loaded with a therapeutic or diagnostic agent, the methodcomprising, recombinantly expressing mutated human papillomavirus (HPV)L1 capsid protein mutant and wild-type HPV L2 capsid protein inbacterial cells; isolating mutated HPV L1 capsid protein, wild-type HPVL2 capsid protein, mutated HPV L1 capsomeres, mutated HPV L1/wild-typeL2 capsomeres, or a combination thereof; combining the capsid proteinsand/or capsomeres with the agent and reassembly buffer containing saltand HEPES buffer, or salt and Histidine-HCl buffer; and dialyzing thecombination of protein and reassembly buffer to produce HPV nanosphereparticles loaded with the agent. In some embodiments, the reassemblybuffer contains 0.5M NaCl, 5 mM CaCl2, and 40 mM HEPES (pH 6.8), or (2)0.5M NaCl, 5 mM CaCl2, and 40 mM Histidine-HCl (pH 5.2).

In some aspects provided herein are methods of delivering and evaluatinga cancer therapy comprising: (a) identifying a subject with cancer; (b)labeling tumor tropic nanosphere particles; (c) loading the tumor tropicnanosphere particles with a therapeutic agent; (d) administering adetectable amount of the nanosphere particles to the subject; and (e)determining the presence or amount of the nanosphere particles in thesubject during and after a period of a treatment. In some embodiments,the label is selected from the group consisting of a fluorescent label,a radioactive label and a chemiluminescent label.

For some viruses and other pathogens, tropism is recognized as a naturalphenomenon which may be referred to as “host tropism” or “cell tropism”in which tropism refers to the way in which different viruses/pathogenshave evolved to preferentially target specific host species. Forexample, HIV has a glycoprotein (gp120) which recognizes and bindsspecifically to the CD4 surface receptor cells of macrophages and Tcells. In this example, the CD4 receptor cells and other necessarycofactors act as a stimulus in attracting human immunodeficiency virusto the surface of an immune cell. Recombinant Adeno Associated Virus(AAV) virions have been shown to exhibit tropism for respiratoryepithelial cells (Flotte et al. 1992 Am. J. Respir. Cell Mol. Biol.7:349-356).

According to aspects of the invention, NSPs derived from proteins ofcertain viruses described herein exhibit broad tumor tropism alsoreferred to herein as universal tumor tropism. Accordingly, NSPsdescribed herein can be used for targeting one or more tumors with atherapeutic agent, for example in a subject diagnosed as having cancer.In some embodiments, the term “subject” includes animals, such aswarm-blooded mammals, for example, humans and primates; avians; domestichousehold or farm animals such as cats, dogs, sheep, goats, cattle,horses and pigs; laboratory animals such as mice, rats and guinea pigs;fish; reptiles; zoo and wild animals; and the like. Also, in someembodiments, NSPs described herein can be used to detect cancer in asubject by selectively delivering a diagnostic agent (for example adetectable agent, a contrast agent, or other diagnostic agent) to cancertissue. In some embodiments, this can increase the detectability ofcancer tissue or cells and/or enhance the contrast between cancer tissueor cells and surrounding healthy tissue, thereby assisting in thedetection or diagnosis of cancer. It should be appreciated thatdiagnostic reagents described herein can be used alone or in combinationwith other diagnostic procedures to help detect or diagnose cancer.

In some embodiments, NSPs described herein can be used to target anytumor, because the NSPs do not discriminate between different tumorcells (e.g., relative to their preference of tumor cells over non-tumorcells). Contrary to expectations, NSPs described herein (e.g., NSPscontaining mutated HPV L2 capsid proteins) display a strong universaltumor tropism for all types of tumor cells lines tested. In addition, insome embodiments NSPs described herein that are not taken up by solidtumor cells will be innocuously eliminated through normal biologicalroutes. For example, NSPs that lack any viral genetic material or hostcell genetic material will not replicate and will be eliminated withoutdamaging healthy cells.

NSPs of the present invention provide a novel combination of viralproteins that can be assembled with drugs or diagnostic agents, and thathave been shown to display a universal tumor tropism. Surprisingly, insome embodiments, modified NSPs having reduced or altered immunogenicityretain their ability to identify tumors with the same mechanismpreviously described by universal tumor tropism. Accordingly, modifiedNSPs (e.g., modified to reduce or prevent cross-reactivity with hostantibodies, for example HPV antibodies) can be modified to deliverreagents (e.g., as surface modifications, as fusion proteins, and/orencapsulated or mixed within the structure of the NSP) selectively totumor tissue or cells (e.g., to solid tumors). In some embodiments,radioisotopes are chemically attached to the surface of an NSP. In someembodiments, small molecule drugs are chemically attached to the surfaceor interior structure of an NSP. In some embodiments the charge of theinterior or exterior of the NSP is modified with electrolytes to enhancethe electrostatic interaction with the NSP and small molecule drugs. Insome embodiments, siRNA, DNA, or drugs are mixed within the structure ofthe NSP.

NSPs described herein display a significant and surprising tumor tropismfor primary tumors as well as to metastases derived from these tumors,even when metastases are distant from the primary tumor (e.g. brain,bone or lung metastases).

In some embodiments, it has been found surprisingly that an assembledNSP containing Papillomavirus L1 structural proteins but noPapillomavirus L2 structural proteins also displays universal tumortropism. Accordingly, NSPs described herein that are derived fromcombinations of L1 structural proteins with therapeutics (but with no L2protein) can also be used to target tumors.

In some embodiments, provided herein are methods and compositions forproducing virion derived protein nanoparticles containing one or moretherapeutic or diagnostic agents. The nanospheres particles of thepresent invention may be derived based on proteins found naturally inthe Herpes Simplex Virus (HSV), the Respiratory Syncytial Virus (RSV),the Polyoma Virus, the beta papilloma virus (β-HPV), alpha-papillomavirus (α-HPV), non-human Papillomavirus (Bovine Papillomaviurs, MacaquePapillomavirus, Cotton Rabbit Papillomavirus, Murine papillomavirus),Epstein Barr Virus, Parvovirus or the Rotavirus. In some embodiments,methods and compositions for encapsulating an agent within a NSP mayrequire an initial isolation and purification of capsid proteinsproduced in a host cell system (e.g., yeast, mammalian cell, insectcell, E. coli) and subsequent reassembly in vitro. In some embodiments,methods and compositions for encapsulating an agent within a NSP mayrequire an isolation and purification of capsid proteins produced in acell free in vitro expression system such as E. coli lysate.

In some embodiments, methods for preparing an NSP to combine atherapeutic or diagnostic agent with papillomavirus proteins include: 1)total disruption and disassembly followed by loading of therapeutics andreassembly; 2) modifications to open pores with no disassembly followedby drug and closing of the pores; 3) chemical binding of radioisotopesto L1 amino acids; and/or 4) chemical binding of radioisotopes to L1 andL2 amino acids.

In some embodiments, provided herein are in vitro methods ofencapsidating an agent within a virus-like nanosphere particlecomprising: isolating viral capsid proteins directly from a host cell;incubating the capsid proteins with an agent in a reaction volume; andassembling the capsid proteins and agent to form a nanosphere particle,thereby encapsidating the agent within the nanosphere particle. In someembodiments, the capsid proteins comprise mutations that modify theserotype-specific immunogenicity in a subject.

In some embodiments, provided herein are intracellular methods ofencapsidating a nucleic acid within a virus-like nanosphere particlecomprising: providing a host cell that expresses a viral capsid protein;and incubating the host cell with a nucleic acid encoding a nucleic acidunder conditions that promote intracellular nanosphere particleformation, thereby encapsidating the nucleic acid within the nanosphereparticle intracellularly. In some embodiments, the capsid proteinscomprise mutations or modifications that provide a modifiedserotype-specific immunogenicity in a subject that preventscross-reactivity with pre-existing antibodies.

In some embodiments, provided herein are methods of isolating a viralcapsid protein comprising: isolating nuclei or obtaining isolated nucleifrom a preparation of host cells that express the viral capsid protein;sonicating the nuclei; and isolating the capsid proteins from thesonicated nuclei in the form of a monomer, capsomere or oligomer. Insome embodiments, the methods of isolating a viral capsid proteincomprise isolating the soluble fraction from the host cell where themonomer, capsomere or oligomer proteins are present. In someembodiments, the capsid proteins comprise mutations or modificationsthat provide a modified serotype-specific immunogenicity in a subjectthat prevents cross-reactivity with pre-existing antibodies.

In any one of the embodiments described herein, the viral capsid proteinmay be a papillomavirus capsid protein. In some embodiments, thepapillomavirus capsid protein is L1. In some embodiments, thepapillomavirus capsid protein is L2. In some embodiments, isolation ofcapsid proteins may include isolation of only L1 capsid proteins, onlyL2 capsid proteins, or a combination thereof. In some embodiments, thecapsid protein is a new non-naturally found serotype derived from thehuman papillomavirus (HPV) serotype 16/serotype 31 L1 protein (HPV16/31L1*) having mutations that reduces a subject's immunogenicity andantibody cross-reactivity to the protein.

In some embodiments, human papillomavirus capsid proteins are isolatedfrom sonicated nuclei of host cells that express both L1 and L2 capsidproteins (wild-type and/or mutant). In some embodiments, the L1 and L2capsid protein and/or capsomeres are obtained from the soluble fractionof the host cell (e.g. E. coli). In some embodiments, the L1 and L2capsid proteins are independently expressed. That is, for example, L1may be expressed by a first nucleic acid (e.g., plasmid DNA), while L2may be expressed by a second nucleic acid, different from the firstnucleic acid.

In some embodiments, an agent is a therapeutic agent or a diagnosticagent. In some embodiments, the agent is a nucleic acid, such as anexpression modulatory nucleic acid (e.g., long non-coding RNA (lncRNA).In some embodiments the nucleic acid is a microRNA. In some embodimentsthe nucleic acid is a hybrid siRNA. In some embodiments the nucleic acidis an antisense oligonucleotide. In some embodiments, the nucleic acidis a small-interfering RNA (siRNA), short-hairpin RNA (shRNA), or avector encoding an siRNA or shRNA. In certain other embodiments, theagent is a nucleic acid (e.g., DNA) encoding for a biologically activeprotein. In some embodiments, the agent is a small molecule (e.g.taxane, SFU, Gemcitabine).

In some embodiments, provided herein are intracellular methods ofencapsidating an agent within a human papillomavirus-derived nanosphereparticle comprising: providing a host cell that expresses an L1 capsidprotein encoding by a first nucleic acid and an L2 capsid proteinencoded by a second nucleic acid different from the first nucleic acid;and incubating the host cell in the presence of an agent within the cellunder conditions that promote intracellular nanosphere particleformation, thereby encapsidating the agent within the NSPintracellularly. In some embodiments, the L1 capsid protein comprises amutation that reduces a subject's immune response and antibodycross-reactivity to the protein.

Any one of the embodiments described herein may further comprisepurifying an assembled, loaded NSP.

In some embodiments, provided herein are methods of producing avirus-like nanosphere particle in a host cell comprising: expressing anL1 capsid protein from a first nucleic acid in a host cell; expressingL2 capsid protein from a second nucleic acid in the host cell, whereinthe first nucleic acid is separate from the second nucleic acid; andisolating an assembled nanosphere particle from the host cell. In someembodiments, the ratio of L1 protein to L2 protein differs from a normalL1 protein to L2 protein ratio (e.g., different from ratio found innature). In some embodiments, L1 capsid proteins are used without L2capsid proteins.

In some embodiments, provided herein are methods for selectivelydelivering an agent to a tumor in a subject, the method comprisingadministering a tumor tropic nanosphere particle to a subject, whereinthe tumor tropic nanosphere particle is associated with an agent, and isfree of host cell and viral nucleic acid, and wherein the tumor tropicnanosphere particle comprises one or more viral capsid proteins withouta heterologous targeting agent (e.g., without being associated with orattached to an antibody, peptide, ligands, receptor-binding moieties, orother targeting molecule that a capsid protein would otherwise naturallybe associated of attached).

In some embodiments, provided herein are methods for preparing ananosphere particle for selectively delivering a therapeutic ordiagnostic agent to a cancer in a subject, the method comprising, obtainviral capsid proteins without any host or viral nucleic acid, andreassembling the capsid proteins in the presence of a therapeutic ordiagnostic agent.

In some embodiments, provided herein are methods of producing humanpapillomavirus (PV) nanosphere particles loaded with an agentcomprising: recombinantly expressing mutant PV L1 and wild-type PV L2capsid proteins, or mutant PV L1 without any PV L2 capsid proteins invitro in E. coli cells, wherein the mutant PV L1 capsid protein hasmutations that differs from the wt HPV; isolating the L1 and L2capsomeres, or L1 capsomeres; combining the capsid protein capsomereswith the agent and reassembly buffer containing salt and HEPES buffer,or salt and Histidine-HCl; and dialyzing the combination of protein andbuffer to produce HPV nanosphere particles loaded with the agent.

In some embodiments, provided herein are methods for delivering andevaluating a cancer therapy comprising: identifying a subject withcancer; labeling tumor tropic nanosphere particles; loading the tumortropic nanosphere particles with a therapeutic agent; administering adetectable amount of the nanosphere particles to the subject; anddetermining the presence or amount of the nanosphere particles in thesubject during and after a period of a treatment.

In some embodiments, provided herein are compositions for the treatmentor diagnosis of cancer cells, the composition comprising a therapeuticor diagnostic agent formulated with a nanosphere particle, wherein thenanosphere particle comprises structural proteins from HSV, RSV,Polyoma, PV, Epstein Barr or Rotavirus.

In some embodiments, provided herein are compositions for the treatmentor diagnosis of cancer cells, the composition comprising a therapeuticor diagnostic agent formulated with a nanosphere particle, wherein thenanosphere particle comprises a mutated or modified PV L1 protein,wherein the mutation or modification reduces or modifies the PV serotypespecific immunogenicity of the nanosphere particle.

These and other embodiments are described in more detail in thefollowing detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows production of Pseudovirions (PsV) in 293TT cells

FIG. 2A shows capsomere particle production and purification method inE. coli and further loading and reassembly of Nanosphere Particles(NSPs) in vitro. FIG. 2B shows capsomere particle production andpurification method in E. coli, in vitro reassembly into NSPs andfurther labeling with radioisotopes.

FIG. 3A shows western blot with L1 and L2 expression after isolation ofsoluble fraction from E. coli. FIG. 3B shows electron microscopic imagesdisplaying spontaneously reassembled particles after reducing agent(DTT) concentration was lowered and salt (sodium chloride) concentrationwas increased. L1/L2 capsomeres from E. coli homogenate were purified byaffinity chromatography on a heparin column.

FIG. 4A shows NSPs production method in 293TT cell line. FIG. 4B showsNSPs production method in 293TT cell line and further labeling withradioisotopes.

FIGS. 5A-5B shows treatment of mice bearing a metastatic orthotopicovarian tumor derived from SKOV-3 cells with NSPs and biodistributioncomparison between tumor bearing and non-bearing animals. FIG. 5A showsresults show in vivo bioluminescent signals as taken 48 hours afterdosing. A single injection was administered with NSPs-luc (0.65 ml) whentumor size reached medium to large by palpitation on day 77 post tumorimplantation. FIG. 5B shows results of ex vivo tissue bioluminescentimaging of the primary tumor and tumors metastasized to the lung, theliver, the spleen GI-LN at 48 hours post dosing. Primary tumors anddistant metastatic sites including lung, spleen, liver, mesenteric lymphnodes, femur and brain were dissected and imaged ex vivo forbioluminescence.

FIGS. 6A-6E show the treatment of mice bearing a metastatic orthotopicovarian tumor derived from SKOV-3 cells with NSPs and PsV to compare thetumor tropism of the two types of particles. Graphs show a comparisonbetween the biodistribution of NSPs and PSV particles delivering aluciferase reporter gene in an SKOV-3 ovarian orthotopic model. FIG. 6Ashows a comparison of NSPs and PsV biodistribution found in the lungs inan ovarian orthotopic SKOV-3 model as determined by a luminescentreporter gene. FIG. 6B shows a comparison of NSPs and PsVbiodistribution found in the liver in an ovarian orthotopic SKOV-3 modelas determined by a luminescent reporter gene. Results show that bothtypes of particles have a similar tropism both to the primary tumor(FIG. 6A) and to the distant metastases (FIG. 6E). This resultdemonstrate that NSPs containing mutant L1/L2 particles retain the tumortropism observed with wild type PsV.

FIG. 7 shows plasmid pL1*L2 DNA sequence encoding mutant L1 (L1*) and L2human codon-optimized (SEQ ID NO:1).

FIGS. 8A-8B show a HPVL1*-mutant DNA sequence (human codon optimized)(SEQ ID NO:2).

FIG. 9 shows HPVL1*-mutant DNA sequence (E. coli optimized) (SEQ IDNO:3).

FIG. 10 shows HPV6L2 DNA sequence (E. coli optimized) (SEQ ID NO:4).

DETAILED DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

In some embodiments, aspects of the invention relate to methods andcompositions for producing virion-derived protein nanosphere particles(NSPs) that contain therapeutic agents or diagnostic agents for deliveryselectively to cancerous cells of a subject without eliciting aserotype-specific immune response in the subject. Surprisingly, humanpapillomavirus (HPV) variants that have reduced immunogenicity retainbroad tumor tropism. Accordingly, in some embodiments, methods andcompositions have been developed for effectively encapsidating atherapeutic or diagnostic agent within NSPs (e.g.,papillomavirus-derived nanosphere particles) that can be used to deliverthe agent selectively, in some instances without a targeting molecule,to cancerous cells of a subject (e.g., a human subject), without harminghealthy, noncancerous cells.

A “nanosphere particle” herein refers to an organized capsid-likestructure comprising self-assembling ordered arrays of one or more viralcapsid proteins that do not include a viral genome. Nanosphere particlesare morphologically and antigenically similar to authentic virions, butthey lack viral genetic material (e.g., viral nucleic acid), renderingthe nanosphere particle non-infectious. Nanosphere particles may beproduced in vivo, in a suitable host cell, such as mammalian, yeast,bacterial, or insect host cell. Nanosphere particles may also beproduced in vitro in a cell free expression system (e.g. E. colilysate).

In some embodiments, nanospheres may be assembled from proteins foundnaturally in herpes simplex virus (HSV), Rous sarcoma virus (RSV),alpha-papilloma virus (α-HPV), beta-papilloma virus (β-HPV), Non-HumanPapillomavirus (Bovine Papillomavirus, Murine Papillomavirus, CottontailRabbit Papillomavirus, Macaque Papillomavirus), Epstein Barr virus,Hepatitis virus, Rotavirus, or other virus or virus-like particles. Insome embodiments, a nanoparticle may be derived from the following:Adenoviridae, Papillomaviridae, Parvoviridae, Herpesviridae, Poxviridae,Hepadnaviridae, Polyomaviridae, Anelloviridae, Reoviridae,Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae,Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, orArteriviridae.

In some embodiments, other virus proteins which may be used as deliveryagents within the scope of the present invention are not limited to butmay include: retroviruses, adenoviruses, adeno-associated viruses,lentiviruses, poxivurses, baculoviruses, and bacteriophages. Otherviruses that are not tumor tropic can be modified by adding a targetmolecule to its structure.

In some embodiments, nanospheres are prepared using a variant capsidprotein having one or more mutations that modify the viralserotype-specific immunogenicity that prevent antibody cross-reactivity.In some embodiments, the term “serotype-specific immunogenicity”includes the ability of a serotype-specific viral antigen or epitope toelicit an immune response (humoral and/or cell-mediated) in a subject. Aserotype refers to a distinct variation within a species of virus (e.g.,human papillomavirus serotype 16 (HPV16) and 31 (HPV31) are differentserotypes based on their cell-surface antigens)

Infection by one or more human papillomavirus high-risk serotypes (e.g.HPV16) is causally associated with cervical cancer (zur Hausen H.,Cancer Res., 1989, 49, 4677-4681). Native virions of HPV arenonenveloped 50- to 60-nm-diameter icosahedral structures composed of 72capsomeres, and each capsomere is composed of five L1 capsid proteins(Baker, T. S., et al., Biophys J., 1991, 60, 1445-56; Trus, B. L., etal., Nat Struct Biol. 1997, 4, 413-20). Native virions of HPV alsoinclude L2 capsid proteins. A “capsid protein” refers to individualcapsid proteins that assemble to form a viral capsid structure. A capsidprotein can be in the form of a single protein monomer, or severalcapsid proteins can form an oligomer (e.g., pentamers, trimers). A“capsomere” refers to an oligomeric configuration of capsid protein. Forexample, capsomeres may comprise at least one L1 capsid protein (e.g., apentamer of L1).

In some embodiments, capsid proteins are expressed intracellularly (in ahost cell) in the presence of an agent to produce a nanosphere particleencapsidating the agent. A nanosphere particle encapsidating an agent isherein referred to as a “loaded” nanosphere particle. In someembodiments, a nucleic acid encoding a capsid protein (e.g., L1 or L2)is introduced into a host cell together with a nucleic acid encoding anagent, for example, a biologically active protein or peptide. In someembodiments, the nucleic acid encoding a capsid protein may encode morethan one capsid protein, e.g., L1 and L2 capsid proteins. In someembodiments, two different capsid proteins are expressed intracellularlyby introducing a first nucleic acid encoding a first capsid protein anda second nucleic acid encoding a second capsid protein such that eachcapsid protein is independently expressed by the cell. Expression ofcapsid protein in the presence of an agent results in intracellularformation (assembly) of nanosphere particles containing (encapsidating)the agent. Loaded nanosphere particles can be isolated directly from thehost cell.

In some embodiments, one or more different capsid proteins are expressedintracellularly in the absence of an agent. In such embodiments, theassembled nanosphere particles are first isolated from the host cell,and then dissociated into individual capsid protein monomers and/orprotein oligomers. These isolated capsid proteins may then bereassembled in the presence of an agent to produce a loaded nanosphereparticle. Direct isolation of individual capsid proteins (rather thanisolation of assembled NSPs) may, in some embodiments, reduce the riskof host cell contamination of the loaded nanosphere particle (e.g., withhost cell nucleic acid, antigens, other exogenous material).

The terms “dissociated” and “disassembled” (used interchangeably) hereinrefers to the deconstruction of viral particles, capsids, or capsomeresinto individual capsid proteins, for example, L1 and/or L2 capsidproteins. L1 and/or L2 capsomeres and/or capsid proteins that areisolated directly from cells can then be used in vitro to encapsidate atherapeutic or diagnostic agent, resulting in a “clean” preparation ofL1 and/or L2 proteins, free of contaminating material (e.g., nucleicacid, antigens, or other material) from the host cell. The term“encapsidate” is also referred to herein as “loading” or“encapsulating,” and refers to the process of surrounding an agent(“payload”) with capsid proteins. The term “encapsulating” and“assembling” may also be used interchangeably herein when referring to amethod of producing a nanosphere particle loaded with an agent. The term“assembling” refers to the process by which capsid proteins associate toform a capsomere, capsid, or nanosphere particle. The term “payload” and“agent” are used interchangeably and refer to any substance encapsidatedwithin or attached to a nanosphere particle, for example, a therapeuticagent or a diagnostic agent.

In some embodiments, L1 and L2 capsid proteins are producedindependently from independent nucleic acids (e.g., different vectors).In some embodiments, they can be produced in the same cell (e.g., usingtwo different vectors within the same cell), or in a different cell(e.g., different host cells of the same type or different types of hostcell). This approach allows the ratio of L1 and L2 proteins to be variedfor either in vitro or intracellular assembly. The term “assembly”refers to the process by which capsid proteins come together to form ananosphere particle. Independent production of L1 and L2 capsid proteinspermits nanosphere particle assembly (e.g., in vitro or intracellularly)with varied ratios of L1 and L2, which may be advantageous during thedelivery process. For example, a higher ratio of L2 to L1 in theassembled structure may result in a nanosphere particle having a highernucleic acid binding affinity and more efficient intracellular delivery.

In some embodiments, L1 capsid proteins are isolated (e.g., from cellsthat express L1 alone, or from cells that express L1 and L2) and usedindependently of L2 capsid proteins. For example, L1 proteins may beassembled to form capsomeres or nanospheres that do not contain L2proteins. In some embodiments, L1 proteins may be assembled (e.g., invivo or in vitro) along with a payload to form a capsomere or nanospherethat encapsulates the payload (e.g., therapeutic or diagnostic agent).In some embodiments, an L1 capsid protein having one or more amino acidchanges (e.g., a sequence alteration or a chemical amino acidmodification such as a PEGylation or other modification) that reduceimmunogenicity is used.

In some embodiments, nanospheres formed by L1 capsid proteins alone(with or without amino acid sequence modifications), for example HPV L1capsid proteins, exhibit universal tropism as described herein and canbe used for cell or tissue-specific delivery of one or more diagnosticor therapeutic agents.

In some embodiments, nanosphere particles or proteins are produced ininsect cells, yeast, bacterial cells, or mammalian cells. In someembodiments, nanosphere particles or proteins are produced in cell-freeexpression systems.

In some embodiments, a nanosphere particle comprises a variant capsidprotein having one or more mutations that reduce or modify HPV serotypespecific immunogenicity. For example, a nanosphere particle may comprisea non-natural variant HPV-16 L1 protein having one or more mutationsthat prevents HPV 16 antibodies to cross-react with its structure.

In some embodiments, methods include delivering a therapeutic ordiagnostic agent to a cancerous cell without delivering the agent to anon-cancerous cell, wherein the delivery vehicle is a nanosphereparticle having reduced or modified serotype-specific immunogenicity. Insome embodiments, methods include delivering a therapeutic agent tocancerous cells present at a single anatomical location or present atmultiple, different anatomical locations, for example, cancerous cells(metastases) present in bone, lung and/or brain. In other embodiments,methods include inhibiting the proliferation of cancerous cells, withoutinhibiting proliferation of non-cancerous cells, wherein the cancerouscells are inhibited by delivery of an inhibitory agent, and wherein thedelivery vehicle used to deliver the inhibitory agent to the cancerouscells is a nanosphere particle having reduced or modifiedserotype-specific immunogenicity. In still other embodiments, methodsinclude detecting the presence of a cancerous cell at a singleanatomical location or at multiple, different anatomical locations (e.g.metastases). In yet other embodiments, methods include monitoring theefficacy of a cancer therapy.

Variant Capsid Proteins Having Reduced or Modified Immunogenicity

In some embodiments, human papillomavirus (HPV) nanosphere particlescomprise L1 capsid proteins, L2 capsid proteins, or both. The proteinsmay be wild-type or modified. In some embodiments, nanosphere particlescomprise a naturally occurring HPV capsid protein and/or a variant HPVcapsid protein having reduced or modified serotype-specificimmunogenicity (e.g., in a subject). For example, in some instance,nanosphere particles comprises L1 and/or L2 proteins that have beenmodified (e.g., mutated, substituted, inserted, or deleted) to reduceimmunogenicity against serotype-specific HPV antibodies. In someembodiments, nanosphere particles comprise L1 and/or L2 variantsequences that are not recognized by existing antibodies against HPV(e.g., HPV16L1), which may be present in subjects who have an HPVinfection or who have received an HPV vaccine. Examples of variantcapsid proteins are described in WO 2010/120266, incorporated herein byreference in its entirety. A capsid protein may have an amino acidvariation that results in reduction or avoidance of neutralization bythe immune system of the subject. In some embodiments, a nanosphereparticle contains a recombinant capsid protein (e.g., a recombinant HPVL1 and/or L2 protein) having an amino acid variation that results inaltered protein immunogenicity in a subject. In some instances, ananosphere particle having such reduced or modified immunogenicity mayalso retain its capability of packaging (loading) and deliveringmolecules/agents to a subject.

In some embodiments, an amino acid of a capsid protein is modified toenhance the positive charge of the nanosphere particle interior. In someembodiments, modifications are introduced to permit a strongerelectrostatic interaction of nucleic acid molecules with an amino acidthat faces the interior of the nanosphere particle, or to promoteretention of the nucleic acid within the nanosphere particle (e.g.,avoid leakage of nucleic acid molecules out of the nanosphere particle).As referenced above, examples of such modifications are described in WO2010/120266. Any “modified” nanosphere particle (that is, any nanosphereparticle containing a modified capsid protein) may be loaded with anagent for selective delivery to a diseased/cancerous cell, sparingnormal cells.

In some embodiments, HPV L1 and/or L2 capsid proteins may be chemicallymodified such that the resulting protein comprises linkers that enablethe binding of small molecules to its internal structure or to itsexternal structure. In some embodiments, HPV L1 and/or L2 proteins maybe fused to other molecules (e.g., lipids, polymers) that containhydrophobic drugs (e.g., taxanes), which may provide additionalfunctionality. In some embodiments, HPV L1 and/or L2 proteins may befused to other proteins, which may provide tissue specific tropism.Examples of such modifications are described, for example, in U.S. Pat.No. 6,991,795, incorporated herein by reference in its entirety. Theseother proteins may be viral or non-viral and may, in some embodiments,be host-specific or cell type-specific. Nanosphere particles maycomprise a recombinant protein or fragment thereof (e.g., an HPV capsidand/or surface protein or fragment thereof). In some embodiments,nanosphere particles may be based on naturally-occurring particles thatare processed to incorporate an agent as described herein. In someembodiments, one or more sequence variants may be used as describedherein.

According to some aspects of the invention, nanosphere particlesdescribed herein exhibit tropism for specific cell types. In someembodiments, cancer cells and cancer metastases are targeted naturallyby the particles without requiring any additional targeting agents. Insome embodiments, cancer stem cells are targeted. Accordingly,nanosphere particles described herein can be administered to a subjectwithout a targeting agent and the nanospheres will preferentiallyaccumulate in tissues that contain tumor cells, tumor metastases, cancerstem cells, or a combination thereof. Accordingly, nanosphere particlesdescribed herein can be used to preferentially deliver therapeuticand/or diagnostic agents to these specific cells sparing other types ofcells. In some embodiments, the tumors are non-mucosal (e.g., innon-mucosal tissue(s)). In some embodiments, the tumors are solidtumors.

According to aspects of the invention, nanosphere particles that containmodified capsid proteins (for example to reduce the immunogenicity ofthe capsid proteins, for example by altering the immunogenicity of theL1 capsid protein) also can retain tumor tropism properties that areuseful for targeted delivery. These particles also have the benefit ofevading detection by the host immune system, thereby increasing theamount or efficiency of targeting and delivery to the target cells ofinterest. According to aspects of the invention, nanosphere particlesthat contain L1 capsid proteins (for example modified L1 capsidproteins), but not L2 capsid proteins also can retain tissue tropismproperties that are useful for targeted delivery.

However, it should be appreciated that other combinations of HPVproteins (e.g., capsid proteins) or peptides may be used herein. In someembodiments, one or more targeting agents (e.g., a targeting peptidefused to a capsid protein) may be used to enhance or alter the tissuetropism of the particles described herein.

In some embodiments, nanospheres that include capsid proteins with oneor more amino acid alterations in a hypervariable or surface exposedloop retain the property of cell-specific or disease-specific targeting.These variant nanospheres can be used for targeted delivery according toaspects of the invention without using any other targeting agents.Examples of conformation-dependent type-specific epitopes that can bemodified to alter immunogenicity are found on the surface of HPVnanosphere particles within hyper-variable loops, where the amino acidsequence is highly divergent between HPV serotypes. These loops aredesignated BC, DE, EF, FG and HI. Many neutralizing antibodies aregenerated against epitopes in these variable loops and aretype-specific, with limited cross-reactivity, cross-neutralization andcross-protection. Different HPV serotypes induce antibodies directed todifferent type-specific epitopes and/or to different loops. In someembodiments, HPV L1 and/or L2 may be mutated at an amino acid positionlocated in a hyper-variable, surface-exposed loop. The mutation may bemade at an amino acid position within a loop that is not conservedbetween HPV serotypes. This position may be completely non-conserved(any amino acid may be at this position), or the position can beconserved.

Independent Expression Vectors

In some embodiments, an expression vector is used to express an HPVcapsid protein, a variant HPV capsid protein, or a combination thereof.For example, in some embodiments, a mutant HPV16L1 protein havingreduced immunogenicity (referred to herein as “L1*”) is co-expressedwith L2 in a host cell system (e.g., Sf9 or 293TT cells). In someembodiments, each protein is expressed by an independent vector (e.g.,L1* is expressed by vector A, while L2 is expressed by vector B).

In some embodiments, L1 and L2 proteins are expressed in a host cellsystem from independent expression vectors (e.g., plasmids) as opposedto both being expressed from the same vector. The expression of L1 andL2 proteins from independent plasmids permits the relative levels of L1and L2 comprised within a nanosphere particle to be optimized forparticular applications. This control of L1 to L2 protein ratio alsopermits optimization of molecular structure for delivery of particularagents. In some embodiments, a variety of nanosphere particle structurescan be produced to conform to the need of the different classes ofagents (e.g., DNA, RNA, small molecule, and large molecule), both interms of electrostatic charge and other functions (e.g., DNA bindingdomains, nanosphere particle inner volume, and/or endosomal releasefunction). For example, nanosphere particles with a higher content of L2protein will be better to bind nucleic acids (L2 contains a DNA bindingdomain), whereas nanosphere particles with a smaller content of L2protein will be better for other small molecules. Nanospheres withdifferent ratios of L1:L2 protein will have different interior volumes,which will permit a higher concentration of drug to be encapsidated. Insome embodiments, the release of agent into the cell may also bemodulated. In some embodiments, structures containing more L2 proteinmay have an increased ability to transfer nucleic acids intracellularly.Different ratios of L1:L2 may be used in any of the embodimentsdescribed herein. In some embodiments, a nanosphere contains L1 protein,but not L2 protein.

In some embodiments, each separate expression nucleic acid encodes an L1protein (but not an L2 protein) or an L2 protein (but not an L1 protein)sequence operably linked to a promoter. In some embodiments, othersuitable regulatory sequences also may be present. The separateexpression nucleic acids may use the same or different promoters and/orother regulatory sequences and/or replication origins, and/or selectablemarkers. In some embodiments, the separate nucleic acids may be vectors(e.g., plasmids, or other independently replicating nucleic acids). Insome embodiments, separate nucleic acids may be independently integratedinto the genome of a host cell (e.g., a first nucleic acid integratedand a second nucleic acid on a vector, or two different nucleic acidsintegrated at different positions). In some embodiments, the relativeexpression levels of L1 and L2 proteins may be different in differentcells, differ using different expression sequences, be independentlyregulated, or a combination thereof.

Host Cell Expression Systems

In some embodiments, a capsid protein (e.g., L1 and/or L2) is expressedin a host cell system. In some embodiments, more than one type ofprotein (e.g., L1 and L2) are expressed in a host cell system. In someembodiments, L1 and L2 are expressed in the same host cell (or system).In some embodiments, L1 and L2, or variations thereof, are expressed indifferent host cells (or systems). Any one of the proteins ornanoparticles described herein may be produced in an insect cell system,a yeast cell system, a bacterial cell system, a mammalian cell system,plant cell system or in a cell free expression system. Examples of hostcell systems to be used herein include, but are not limited to,Spodoptera frugiperda (sf) cells, Escherichia coli cells, and 293T or293TT mammalian cells. In particular embodiments, L1 and/or L2 and/orvariants thereof are expressed intracellularly, where they may formcapsomeric (e.g., oligomeric) structures during cellular growth (e.g.,fermentation). Subsequent to structure formation, in some embodiments,the capsid proteins and/or structures may be isolated from host cellnuclei or from the host soluble fraction. Any suitable method may beused to isolate the nuclei, for example, sonication, or other isolationmethod. After isolation, capsid proteins and/or structures may bepurified by any suitable means, for example, column chromatography, orother purification method. In some embodiments, rather than isolatingthe capsid proteins from cell nuclei, the proteins are permitted toassemble intracellularly in the presence of a payload to form loadednanoparticles.

Directly isolating capsid proteins from nuclei of cells (rather thanisolating assembled nanoparticles) provides several benefits. Forexample, in some embodiments, there is a reduced risk of encapsidatingand transferring genetic information (e.g., DNA, RNA) from the host cellto a subject receiving the loaded nanoparticle. In some embodiments,isolated capsid proteins are assembled in a cell-free system withpayload to produce a nanoparticle loaded with that payload (e.g. DNAcoding for a biologically active protein, small molecule, RNA). Incertain embodiments, de novo assembly of nanoparticles as describedherein (as opposed to using pre-formed nanoparticles) results in alarger percentage of loaded nanoparticles.

In some embodiments, a capsid protein may be expressed recombinantly inany one of the host cell systems described herein.

Non-limiting examples of insect cell systems: in some embodiments, anyone of the capsid proteins described herein may be expressed inSpodoptera frugiperla (Sf) cells, for example, Sf21 cells. In someembodiments, baculoviruses are used to express a gene encoding a capsidprotein, for example, a gene encoding L1 and/or L2 and/or a variantthereof (including recombinant versions). In some embodiments, thecapsid protein is an L1 or L2 protein from a designated serotype ofhuman papillomavirus, for example, HPV16, HPV18, HPV31, HPV33, HPV34,HPV35, HPV52, HPV58, HPV73, and HPV91, and/or as described (Touze etal., FEMS Microbiol. Lett., 2000, 189, 121-7; Touze et al., J. Clin.Microbiol., 1998, 36, 2046-51; and Combita et al., FEMS Microbiol. Lett.2001, 204(1), 183-88). In some embodiments, a gene encoding a capsidprotein is cloned into a plasmid, such as pFastBac1 (Invitrogen). Insome embodiments, insect cells may be maintained in Grace's insectmedium (Invitrogen), or other suitable medium, supplemented with, forexample, 10% fetal calf serum (FCS, Invitrogen), infected withrecombinant baculoviruses, and incubated at 37° C. In some embodiments,cells may be harvest three days post infection, and the nanoparticlepurified. In some embodiments, cells may be re-suspended inphosphate-buffered saline (PBS) containing Nonidet P40 (0.5%), pepstatinA, and leupeptin (1 μg/ml each, Sigma Aldrich), and incubated for 30 minat 4° C. Nuclear lysates may be formed into pellets by centrifugation,re-suspended in ice-cold PBS containing pepstatin A and leupeptin, andthen sonicated. Samples may then be loaded on a cesium-chloride (CsCl)gradient and centrifuged to equilibrium (e.g., 22 h, 27,000 rpm in aSW28 rotor, 4° C.). Cesium-chloride gradient fractions may beinvestigated for density by refractometry and for the presence of L1/L2protein by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamidegel (SDS-PAGE) and Coomassie blue staining. Positive fractions may bepooled, diluted in PBS and pelleted (e.g., in a Beckman SW 28 rotor (3h, 28,000 rpm, 4° C.)). After centrifugation, nanoparticles may bere-suspended in 0.15 mol/L NaCl and sonicated, for example, by one5-second burst at 60% maximum power. Total protein content may bedetermined. Other techniques may be used, as embodiments of theinvention are not limited by these examples.

Non-limiting examples of yeast cell systems: in some embodiments, anyone of the capsid proteins described herein may be expressed in yeastcells. In some embodiments, capsid proteins may be expressed using agalactose-inducible Saccharomyces cerevisiae expression system. Forexample, leucine-free selective culture medium may be used for thepropagation of yeast cultures, and yeast may be induced with mediumcontaining glucose and galactose. Cells may be harvested using anyfiltration means. After resuspension, in some embodiments, cells may betreated with Benzonase and mechanically disrupted (e.g., using ahomogenizer). Cell lysate may then be clarified using any filtrationmeans. An exemplary protocol can be found in Cook et al. ProteinExpression and Purification, 1999, 17, 477-84. Other techniques may beused, as embodiments of the invention are not limited by these examples.

Non-limiting examples of mammalian cell system: in some embodiments, anyone of the capsid proteins described herein may be expressed inmammalian cell systems. Buck et al. (J. Virol. 2004, 78, 751-757)reported the production of papilloma virus-like particles and celldifferentiation-independent encapsidation of genes into bovinepapillomavirus (BPV) L1 and L2 capsid proteins expressed in transientlytransfected 293TT human embryonic kidney cells, which stably expressSV40 large T antigen to enhance replication of SV40 origin-containingplasmids. Pyeon et al. reported a transient transfection method thatachieved the successful and efficient packaging of full-length HPVgenomes into HPV16 capsids to generate virus particles (PNAS, 2005, 102,9311-16). Transiently transfected cells (e.g., 293 cells, for example293T or 293TT cells) may be lysed by adding Brij 58 or similar nonionicpolyoxyethylene surfactant detergent, followed by benzonase andexonuclease V, and incubating at 37° C. for 24 h to remove unpackagedcellular and viral DNA and to allow nanoparticle maturation. The lysatemay then be incubated on ice with 5 M NaCl (to a final concentration of0.8M NaCl) and clarified by centrifugation, or other clearing means.Nanoparticles may be collected by high-speed centrifugation, or othercollection means. Other techniques may be used, as embodiments of theinvention are not limited by these examples.

Non-limiting examples of bacterial cell system: in some embodiments, anyone of the capsid proteins described herein may be expressed inEscherichia coli (E. coli) cells. In E. coli, in some embodiments, apotential contaminant of protein is endotoxin, a lipopolysaccharide(LPS) that is a major component of the outer membrane of Gram-negativebacteria (Schädlich, et al. Vaccine, 2009, 27, 1511-22). In someembodiments, transformed BL21 bacteria may be grown in lysogeny broth(LB) medium containing, for example, 1 mM ampicillin and incubated withshaking at 200 rpm at 37° C. In some embodiments, at an optical density(OD600) of 0.3-0.5 nm, bacteria may be cooled, and Isopropylβ-D-1-thiogalactopyranoside (IPTG) may be added to induce proteinexpression. In some embodiments, after 16-18 hours, bacteria may beharvested by centrifugation, or other harvesting means. Bacteria may belysed by homogenizing, lysates may be cleared, capsid proteins purified,and LPS contamination removed using, for example, chromatographicmethods, such as affinity chromatography or size exclusionchromatography, or other purification methods. Lipopolysaccharidecontamination may also be removed using, for example, 1% Triton X-114.Other techniques may be used, as embodiments of the invention are notlimited by these examples.

With reference now to FIG. 1A, a particle production method fornanospheres with will now be discussed. Prepare plasmid DNA expressingthe capsid proteins of HPV16 L1 and L2 together or on separate vectors121 and transform into bacteria 123. Over 24 hours the L1 and L2proteins will be produced in the cell and will assemble into capsomeres125. Next, harvest, lyse, nuclease digest; purifying using sucrosegradient and Heparin column 127 (or size-exclusion chromatography, ionexchange chromatography, di-filtration, or affinity chromatography).Collect purified capsomeres 129. Reassemble purified particles with“payload” 131. A “payload” may include at least one or a combination ofthe following: DNA, siRNA, micro RNA, antisense oligonucleotide, smallmolecule drug, dye or radioisotope. Column purify to remove free payload133.

With reference now to FIG. 2B, a particle production method fornanospheres will now be discussed. A recombinant DNA molecule containinga sequence encoding a papillomavirus L1 protein or a papillomavirus L2protein or a combination of L1 and L2 proteins is manufactured 221 andthen transfected into a 293TT cell line 223. Preferably, the NSP mayexpress papillomavirus L1 protein or L2 protein or a combination of L1and L2 proteins in the host cell. Over 48 hours the L1 and L2 proteinswill be produced in the cell and will assemble into capsomeres and thenfully assembled VLPs 225. Next, harvest, lyse, nuclease digest;purifying using sucrose gradient and Heparin column 227 (orsize-exclusion chromatography, ion exchange chromatography,di-filtration, or affinity chromatography). Collect purified emptyvirus-like particles 229. Disassembly into capsomeres 231. Reassemblepurified particles with “payload” 233. A “payload” may include at leastone or a combination of the following: DNA, siRNA, drug, dye orradioisotope. Column purify to remove free payload 235.

With reference now to FIG. 3A, a particle production method for radiolabeled nanospheres will now be discussed. Prepare plasmid DNAexpressing the capsid proteins of HPV16 L1 and L2 together or onseparate vectors 321 and transform into bacteria 323. Over 24 hours theL1 and L2 proteins will be expressed in the cell and will assemble intocapsomeres 325. Next, harvest, lyse, nuclease digest; purify usingsucrose gradient and Heparin column 327 or size-exclusionchromatography, ion exchange chromatography, di-filtration, or affinitychromatography. Collect purified capsomeres 329. Reassemble into emptynanosphere particles 331. Label particles with radioactive isotope 333.Column purify to remove free isotopes and enrich for radiolabelednanosphere particles 335.

With reference now to FIG. 3B, a particle production method fornanospheres will now be discussed. A plasmid expressing the capsidproteins of HPV-L1 and L2 421 and transfect into a 293TT cell line 423.Over 48 hours the L1 and L2 proteins will be produced in the cell andwill self-assemble into empty virus-like particles 425. Next, harvest,detergent lyse, nuclease digest, and gradient purify and Heparin column427 (or size-exclusion chromatography, ion exchange chromatography,di-filtration, or affinity chromatography). Collect purified emptyvirus-like particles 429. Label particles with radioactive isotope 431.Column purify to remove free isotopes and enrich for radiolabelednanosphere particles 433.

A single potency curve on the basis of internalized immunotoxin hasshown a sharp reduction in viability once cells internalized >5×106molecules (Pirie et al. (2011) The Journal Of Biological Chemistry, Vol.286, No. 6, pp 4165-4172). Accordingly, according to one aspect of thepresent invention, the production of the immunotoxins to a level offifty percent cytotoxicity is a function of the time for proliferatingtumor cell to uptake plasmid DNA, replicate, transcribe and translateand produce enough immunotoxin to reach levels of at least 50%cytotoxicity as a function of time.

Loading with Therapeutic Agents and/or Diagnostic Agents

Some embodiments described herein relate to methods for producingnanosphere particles having (encapsidating) a medical, a therapeutic, ora diagnostic agent, or a combination thereof.

In some embodiments, a nanosphere particle loaded with an agent may bedelivered to a diseased cell/tissue (e.g., cancerous cell). In someembodiments, a tissue may have pre-malignant disease (e.g., cervicaldysplasia, bronchopulmonary dysplasia, prostate intraepithelialneoplasia). In some embodiments the pre-malignant diseased tissue may beinfected with a virus, such as HPV or HSV. In some embodiments,nanosphere particles may be used to deliver therapeutic agents to treatpre-malignant disease or condition, and/or may be used to deliverdiagnostic agents to diagnose pre-malignant diseases or conditions. Forexample, fluorophores, quantum dots, metals, radioisotopes and/or otherimaging agents may be loaded into nanosphere particles and delivered tothe pre-malignant cells of a subject. In some embodiments, agents may beused to track early stage diseases (e.g., early stage lung cancer, earlystage prostate cancer). Any suitable therapeutic, diagnostic and/orother medical agent may be loaded into the nanosphere particle accordingto the methods described herein and subsequently delivered to a subject.Examples of methods for the administration of nanosphere particles tosubjects are described for example in U.S. Pat. No. 7,205,126,incorporated herein by reference in its entirety.

After isolation of L1 and L2 capsid proteins which may be in the form ofmonomers or oligomers (e.g., pentamers), nanosphere particles may beassembled and loaded, as described herein.

Loading of a nanosphere particle with an agent utilizing a‘disassembly/reassembly’ method has been described, for example, in U.S.Pat. No. 6,416,945 and International Publication No. WO 2010/120266,each incorporated herein by reference. Any one of the previouslydescribed loading methods or methods described herein may be used toencapsidate a therapeutic agent, for example a gene encoding atherapeutic protein, or a diagnostic agent. However, it should beappreciated that any suitable method may be used as aspects of theinvention are not limited in this respect.

In some embodiments, a loading method comprises incubating a nanosphereparticle in a buffer of ethylene glycol tetraacetic acid (EGTA) anddithiothreitol (DTT). Under this condition, a nanosphere particlecompletely disaggregated into monomeric and/or olgomeric capsid proteinstructures. A diagnostic or therapeutic agent as described herein maythen be combined with the disaggregated capsid protein structures, andthen diluted in a buffer of dimethyl sulfoxide (DMSO) and calciumchloride (CaCl2) with or without zinc chloride (ZnCl2) in order toreassemble the capsid proteins into a nanosphere particle, therebyencapsidating the agent. In some embodiments, the presence of ZnCl2increases the reassembly of capsid proteins into a nanosphere particle.Other salts useful in aiding disassembly/reassembly of viral capsidproteins into nanosphere particles are those that include, for example,zinc (Zn), copper (Cu), nickel (Ni), ruthenium (Ru), and iron (Fe).Other salts may also be used in the embodiments described herein.

In some embodiments, loading of a nanosphere particle does not requirean initial nanosphere particle disassembly step.

In some embodiments, the efficacy of nanosphere particle loading anddelivery to a cell may depend, at least in part, on the particular ratioof (a) capsid protein to reaction volume, (b) therapeutic or diagnosticagent (e.g., therapeutic protein) to capsid protein, (c) therapeutic ordiagnostic agent to reaction volume, or other ratio of components. Ananosphere particle loaded with an agent, using a method describedherein, in some embodiments, effectively delivers the agent to at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, 96%, 97%, 98%, 99%, or 99.9% of target cells (forexample, when measured in a cell-based assay).

In some embodiments, a nanosphere particle may be loaded with an agentusing a method comprising: (a) contacting a preparation of capsidproteins with the agent in a reaction volume, wherein (i) the ratio ofcapsid protein to reaction volume may range from 0.01 μg capsid proteinper 1 μl reaction volume to 0.1 μg capsid protein per 1 μl reactionvolume, or from 0.1 μg capsid protein per 1 μl reaction volume to 1 μgcapsid protein per 1 μl reaction volume; (ii) the ratio of agent tocapsid protein may range from 0.01 μg agent per 1 μg capsid protein to0.1 μg agent per 1 μg capsid protein, or from 0.1 μg agent per 1 μgcapsid protein to 10 μg agent per 1 μg capsid protein, and/or (iii) theratio of agent to reaction volume may range from 0.001 μg agent per 1 μlreaction volume to 1 μg agent per 1 μl reaction volume, or from 0.01 μgagent per 1 μl reaction volume to 10 μg agent per 1 μl reaction volume;and (b) reassembling the capsid proteins to form a nanosphere particle,thereby encapsidating the agent within the nanosphere particle. In someembodiments, the ratio of capsid protein to reaction volume ranges from0.2 μg capsid protein per 1 μl reaction volume to 0.6 μg capsid proteinper 1 μl reaction volume; the ratio of nucleic acid to capsid proteinranges from 0.5 μg agent per 1 μg capsid protein to 3.5 μg agent per 1μg capsid protein; and the ratio of agent to reaction volume ranges from0.2 μg agent per 1 μl reaction volume to 3 μg agent per 1 μl reactionvolume.

In some embodiments, dissociating a nanosphere particle or capsidprotein oligomers can be performed in a solution comprisingethylenediaminetetraacetic acid (EDTA) and/or ethylene glycoltetraacetic acid (EGTA) and dithiothreitol (DTT), wherein theconcentration of EDTA and/or EGTA ranges from 0.3 mM to 30 mM and theconcentration of DTT ranges from 2 mM to 200 mM. In certain embodiments,the concentration of EDTA and/or EGTA ranges from 1 mM to 5 mM. Incertain embodiments, the concentration of DTT ranges from 5 mM to 50 mM.Other reagents, volumes, and concentrations may be used, as embodimentsof the invention are not limited by these examples.

The step of reassembling capsid proteins into a nanosphere particle maybe carried out in a solution comprising dimethyl sulfoxide (DMSO), CaCl2and ZnCl2, wherein the concentration of DMSO ranges from 0.03% to 3%volume/volume, the concentration of CaCl2 ranges from 0.2 mM to 20 mM,and the concentration of ZnCl2 ranges from 0.5 μM to 50 μM. In someembodiments, the concentration of DMSO ranges from 0.1% to 1%volume/volume. In some embodiments, the concentration of ZnCl2 rangesfrom 1 μM to 20 μM. In some embodiments, the concentration of CaCl2ranges from 1 mM to 10 mM.

In some embodiments, the loading method is further modified to stabilizethe nanosphere particle, in that the loading reaction is dialyzedagainst hypertonic NaCl solution (e.g., using a NaCl concentration ofabout 500 mM) instead of phosphate-buffered saline (PBS), as waspreviously described. Surprisingly, this reduces the tendency of theloaded nanosphere particle to form larger agglomerates and precipitate.In some embodiments, the concentration of NaCl ranges between 5 mM and 5M. In some embodiments, the concentration of NaCl ranges between 20 mMand 1 M.

In some embodiments, the methods described herein may be used toencapsulate nucleic acids, for example, RNA interference (RNAi) nucleicacids. In some embodiments, short interfering RNA (siRNA), a shorthairpin RNA (shRNA), microRNA, long non coding RNA (lncRNA), hybridDNA-RNA or an antisense nucleic acid (DNA or RNA) is transfected intohost cells (e.g., 293 cells or other mammalian or insect host cells)during the production of the nanosphere particles. Accordingly, loadednanosphere particles may be produced intracellularly to later provideefficient gene silencing effects when delivered to a cell of a subject.In some embodiments, a plasmid is used to express the nucleic acidmolecule. In some embodiments, the plasmid may be ˜2 kilobases (kb) to˜6 kb in size, or larger. In some embodiments, smaller plasmids may beused. In some embodiments, a nanosphere particle encapsidates a plasmidthat is designed to be expressed (and ultimately functional) within atarget cell, for example, a cancerous cell of a subject to which thenanosphere particle is selectively delivered. Consequently, thesilencing nucleic acid encoded by the plasmid will be active within thetarget cell, resulting in knockdown of the targeted gene.

In some embodiments, the therapeutic agent is an inducer of RNAinterference or other inducer of gene silencing. An inducer of RNAinterference may be a siRNA, a shRNA, a hybrid nucleic acid moleculecomprising a first part that comprises a duplex ribonucleic acid (RNA)molecule and a second part that comprises a single strandeddeoxyribonucleic acid (DNA) molecule, a longer double-stranded RNA or aDNA construct for expression of siRNA or longer RNA sequences. Otherinducers or modulators of gene expression include inducers of DNAmethylation, or ribozymes, or aptamers. In other embodiments, thetherapeutic agent can be a modulator of gene expression such as a PNA(Peptide Nucleic Acid).

RNA interference (RNAi) is a process whereby the introduction ofdouble-stranded RNA (dsRNA) into a cell inhibits gene expressionpost-transcriptionally, in a sequence dependent fashion. RNAi can bemediated by short (for example 19-25 nucleotides) dsRNAs or smallinterfering RNAs (siRNA). Double-stranded RNA is cleaved in the cell tocreate siRNAs that are incorporated into an RNA-induced silencingcomplex (RISC), guiding the complex to a homologous endogenous mRNA,cleaving the mRNA transcript, and resulting in the destruction of themRNA.

To induce RNA interference in a cell, dsRNA may be introduced into thecell as an isolated nucleic acid fragment or via a transgene, plasmid,or virus. In some embodiments, nanosphere particles are used to deliverdsRNA to the target cells.

In some embodiments, a short hairpin RNA molecule (shRNA) is expressedin the cell. A shRNA comprises short inverted repeats separated by asmall loop sequence. One inverted repeat is complimentary to the genetarget. The shRNA is then processed into an siRNA which degrades thetarget gene mRNA. shRNAs can be produced within a cell with a DNAconstruct encoding the shRNA sequence under control of a RNA polymeraseIII promoter, such as the human H1, U6 or 7SK promoter. Alternatively,the shRNA may be synthesized exogenously and introduced directly intothe cell, for example through nanosphere particle delivery. In someembodiments, the shRNA sequence is between 40 and 100 bases in length orbetween 40 and 70 bases in length. The stem of the hairpin are, forexample, between 19 and 30 base pairs in length. The stem may containG-U pairings to stabilize the hairpin structure.

Short-interfering RNA (siRNA) sequences are selected on the basis oftheir homology to the target gene. Homology between two nucleotidesequences may be determined using a variety of programs including theBLAST program (Altschul et al. J. Mol. Biol., 1990, 215, 403-10), orBestFit (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA,Wisconsin 53711). Sequence comparisons may be made using FASTA and FASTP(see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98). Toolsfor design and quality of siRNAs, shRNAs and/or miRNAs are known in theart. Web-based online software system for designing siRNA sequences andscrambled siRNA sequences are for example siDirect, siSearch, SEQ2SVM,Deqor, siRNA Wizard (InvivoGen). The specificity can be predicted usingfor example SpecificityServer, miRacle. Target sequences can beresearched for example at HuSiDa (Human siRNA Database), and siRNAdb (adatabase of siRNA sequences). Sequence comparison may be made over thefull length of the relevant sequence, or may more preferably be over acontiguous sequence of about or 10, 15, 20, 25 or 30 bases. In someembodiments, the degree of homology between the siRNA and the targetgene is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99%, or 100%. The siRNA may be between 10bp and 30 bp in length, or between 20 bp and 25 bp, or the siRNA is 20,21 or 22 bp in length.

Short-interfering RNA molecules may be synthesized using standard solidor solution phase synthesis techniques which are known in the art.

In some embodiments, the siRNA has an overhang at one or both ends ofone or more deoxythymidine bases to increase the stability of the siRNAwithin cells by reducing its susceptibility to degradation by nucleases.

In some embodiments, the siRNA is a hybrid nucleic acid moleculecomprising a first part that comprises a duplex ribonucleic acid (RNA)molecule and a second part that comprises a single strandeddeoxyribonucleic acid (DNA) molecule. Targets for the RNA interferencewould include disease causing genes e.g. oncogenes, inflammatory genes,regulatory genes, metabolic genes, viral genes. In one embodiments ofthe invention the target genes would be E6, E7, p53, Sirt-1, survivin,EGFR, VEGFR, VEGF, CTNNB1 or other oncogenes.

Linkages between nucleotides may be phosphodiester bonds oralternatives, for example, linking groups of the formula P (O) S,(thioate); P (S) S, (dithioate); P (O) NR′2; P (0) R′; P (0) OR6; CO; orCONR′2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl(1-9C) is joined to adjacent nucleotides through-O-or-S—.

Modified nucleotide bases can be used in addition to the naturallyoccurring bases. For example, modified bases may increase the stabilityof the siRNA molecule, thereby reducing the amount required forsilencing. The term modified nucleotide base encompasses nucleotideswith a covalently modified base and/or sugar. For example, modifiednucleotides include nucleotides having sugars which are covalentlyattached to low molecular weight organic groups other than a hydroxylgroup at the 3′ position and other than a phosphate group at the5′position. Thus modified nucleotides may also include 2′substitutedsugars such as 2′-0-methyl-; 2-0-alkyl; 2-0-allyl; 2′-S-alkyl;2′-S-allyl; 2′-fluoro-; 2′-halo or 2; azido-ribose, carbocyclic sugaranalogues a-anomeric sugars; epimeric sugars such as arabinose, xylosesor lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purinesand pyrimidines, acylated purines and pyrimidines, and otherheterocycles. These classes of pyrimidines and purines are known in theart and include pseudoisocytosine, N4, N4-ethanocytosine,8-hydroxy-N6-methyladenine, 4-acetyl cytosine, 5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carb oxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine,2methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl cytosine,N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyamino methyl-2-thiouracil, -D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5methoxyuracil, 2methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester,psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester,uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil,5-propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil,5-pentyluracil, 5-pentyl cytosine, and 2,6, diaminopurine,methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

In some embodiments, siRNA molecules or longer dsRNA molecules may bemade recombinantly by transcription of a nucleic acid sequence, forexample contained within a vector as described herein. The vector may beany RNA or DNA vector.

In some embodiments, the vector can be an expression vector, wherein thenucleotide sequence is operably linked to a promoter compatible with thecell. Promoters suitable for use in various vertebrate systems are wellknown in the art. For example, suitable promoters include viralpromoters such as mammalian retrovirus or DNA virus promoters, e.g.,MLV, CMV, RSV, SV40 IEP (immediate early promoter) and adenoviruspromoters and metallothionein promoter. Strong mammalian promoters mayalso be used. It will be appreciated that variants of such promotersretaining substantially similar transcriptional activities may also beused.

In some embodiments, the vector may have at least two promoters, one todirect expression of the sense strand and one to direct expression ofthe antisense strand of the dsRNA. In other embodiments, two vectors maybe used, one for the sense strand and one for the antisense strand.Alternatively the vector may encode RNAs which form stem-loop structureswhich are subsequently cleaved by the cell to produce dsRNA.

The nucleic acid construct may contain a specific cellular, viral orother promoter or repressor of gene expression. The promoter orrepressor may be designed to reflect the context of the cell into whichthe construct is introduced. For example, the construct may contain aviral promoter so expression from the construct is dependent upon thepresence of a viral protein, so that the construct is expressed only inviral-infected cells. Similarly, the construct may have a promoter orrepressor specific to certain cell types or to certain developmentalstages. For example, where the vector is for use in virally infectedcell such as cells infected with HPV, a viral promoter which matches thedisease-causing virus should be used, e.g., a HPV promoter (such as thepromoter causing expression of HPV E6/E7) for HPV-infected cells. Insuch embodiments, the vector will only be expressed in thevirally-infected cells.

Nucleic acids are highly charged and do not cross cell membranes by freediffusion. The hydrophilic character and anionic backbone of nucleicacids such as, for example, siRNAs reduces their uptake by the cells. Insome embodiments, nucleic acids (e.g., siRNA) may be loaded intonanosphere particle (e.g., HPV-nanosphere particle) to efficientlydeliver them to a subject through administration of nanosphere particle.In some embodiments, encapsulating the nucleic acid into a nanosphereparticle increases cellular uptake, allows traversal of biologicalmembrane barriers in vivo, and/or increases the bioavailability of thenucleic acid (e.g., siRNA).

In some embodiments, the agent loaded into the nanosphere particle is ananti-viral agent. In some embodiments, the agent is an anticancer agent.In some embodiments, the anticancer agent is a taxane and/or a platinum(e.g., cisplatinum, carboplatinum, oxaliplatinum).

In some embodiments, the methods described herein may be used toencapsidate radioisotopes or radionuclides. In some embodiments, NSPsencapsidating such radioisotopes or radionuclides may be used to treatcancerous cells. Examples of radioisotopes that may be used with themethods described herein include, but are not limited to, lutetium-177(prepared from ytterbium-176, which is irradiated to become Yb-177,which decays rapidly to Lu-177), yttrium-90, iodine-131, phosphorus-32,boron-10, actinium-225, ismuth-213, lead-212, bismuth-212, polonium-212,thallium-208, Pb-208.

In some embodiments, the methods described herein may be used toencapsidate small molecules or large molecules such as, for example,biologics, oncolytic viral proteins, or a toxic agent, inducers of DNAmethylation, recombinant DNA, ribosomes, aptamers, modulators of geneexpression, proteins, antibodies, siRNA or antisense molecules,biological therapies, viral gene cassettes such as the myc-gene, viralproteins such as the P30 retrovirus protein, or oncolytic viralproteins. Other small or large molecules may be encapsidated. In someembodiments, two or more therapeutic agents may be encapsidated.

In some embodiments, radioisotopes are useful to treat cancer by killingcancer cells (for example by inducing apoptosis). In some embodiments,compositions and methods of the invention can be used for selectivelytargeting radioisotopes to cancer cells. Nanospheres described hereincan be loaded with one or more radioisotopes and administeredsystemically to a subject (for example a subject having one or moreindicia of cancer). Due to the tropism of the nanosphere theradioisotope(s) will be delivered selectively to the cancer cells, andwill be taken up and directed to the cell nuclei, where the effect ofapoptosis will be maximized.

In some embodiments, the therapeutic agent that may be loaded into ananosphere particle using the methods described herein is achemotherapeutic agent, for instance, methotrexate, vincristine,adriamycin, cisplatin, carboplatin, oxaliplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, paclitaxel, docetaxel, fragyline, MeglamineGLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RASfamesyl transferase inhibitor, famesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum,UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776 C85/5FUenhancer, Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine,Alimta/Pemetrexed, ZD 0473/Anormed, YM 116, iodine seeds, CDK4 and CDK2inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide,Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin,Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guaninearabinoside, Taxane Analog, nitrosoureas, alkylating agents such asmelphelan and cyclophosphamide, Aminoglutethimide, Asparaginase,Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin,Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP16-213),Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p′-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′ deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) or Vindesine sulfate, but itis not so limited.

In some embodiments, the therapeutic agent that may be loaded into ananosphere particle using the methods described herein is animmunotherapeutic agent, for instance, Ributaxin, Herceptin, Quadramet,Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex,Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94,anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE,Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide,CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2,MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab or ImmuRAIT-CEA, but itis not so limited.

In some embodiments, the therapeutic agent that may be loaded into ananosphere particle using the methods described herein is an antiviralagent. Examples of anti-viral agents are: Polysulfates (PVAS),Polysulfonates (PVS), Polycarboxylates, Polyoxometalates, Chicoric acid,zintevir, cosalane derivatives, Bicyclams (i.e., AMD3100), T-22, T-134,ALX-40-4C, CGP-64222, TAK-779, AZT (azidothymidine), ddl, ddC, d4T (didehydrodideoxythymidine), 3TC (3′-thiadideoxycytidine), ABC, and otherddN (2′,3′-dideoxynucleoside) analogs, Nevirapine, delavirdine,efavirenz, emivirine (MKC-442), capravirine, thiocarboxanilide UC-781,acyclovir, valaciclovir, penciclovir, famciclovir,bromovinyldeoxyuridine (BVDU, brivudin), Cidofovir, Adefovir dipivoxil,Tenofovir disoproxil, Ribavirin, valacyclovir, gancyclovir, formivirsen,foscarnet, EICAR (5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide),Mycophenolic acid, Neplanocin A, 3-deazaneplanocin A,6′-C-methylneplanocin A, DHCeA(9-(trans-2′,trans-3′-dihydroxycyclopent-4′-enyl)adenine), or c3DHCeA(9-(trans-2′,trans-3′-dihydroxycyclopent-4′-enyl)-3-deazaadenine), asdescribed, for example, in De Clercq, J. Pharmacol. Exp. Ther., 2001,297, 1-10, incorporated by reference herein, but it is not so limited.

In some embodiments, a diagnostic agent may be loaded into a nanosphereusing methods described herein. A diagnostic agent may be a detectablemoiety (e.g., a radioisotope, a fluorescent marker, a radio-opaquemoiety, or other detectable moiety) or a molecule attached to adetectable moiety (e.g., a molecule attached to a radioisotope, referredto herein as a radiolabeled molecule). In some embodiments, a diagnosticagent may be a labeled antibody, for example an antibody that bindsspecifically to a disease antigen (for example a cancer antigen). Insome embodiments, a diagnostic agent may be a labeled receptor bindingmolecule, a labeled ligand or other labeled binding molecule. In someembodiments, a diagnostic agent may be a labeled enzyme or an enzymesubstrate. In some embodiments, a diagnostic agent may be a labelnucleic acid, protein, lipid, carbohydrate or other molecule.

In some embodiments, one or more therapeutic or diagnostic agents areencapsulated within a nanosphere. However, in some embodiments, one ormore therapeutic or diagnostic agents may be attached to the surface ofa nanosphere (for example using a covalent linkage, reversible linkageor an electrolyte solution). In some embodiments, a nanosphere mayinclude both one or more encapsulated and one or more surface boundagents.

Universal Tumor Tropism

In some embodiments, nanosphere particles of the present inventionexhibit universal tumor tropism. Tropism refers to the specificity of apathogen (e.g., virus) for a host tissue and is a natural phenomenon.Typically, pathogens confer tropism for a particular tissue or celltype, for example, human immunodeficiency virus confers tropism forparticular macrophage cells and T cells, while recombinantadeno-associated virus confers tropism for respiratory epithelial cells(Flotte, et al. Am. J. Respir. Cell Mol. Biol., 1992, 7, 349-356).Virion-derived nanosphere particles described herein can confer tropismfor more than one, and in some instances many, different types of cancercells. The term “universal tropism” refers to the nanosphere particle'sspecificity for multiple types of cancer cells, for example, breast,ovarian, lung, and bone cancer cells. Other examples of primary tumorsthat may be targeted or treated using the nanospheres described hereininclude, but are not limited to, prostate tumors, colon tumors,colo-rectal tumors, ovarian tumors, head and neck tumors, liver tumors,pancreatic tumors, renal, and brain tumors. In some embodiments, thetumor is a solid tumor. In some embodiments the tumor is a hematopoietictumor. In some embodiments, the tumor is a primary tumor. In someembodiments, the tumor has metastasized and the nanoparticles can detectthe distant metastases. In some embodiments the nanoparticles may detectthe cancer stem cells within the primary tumor and the metastases.

As used herein, the term “tumor” refers to a tissue comprisingtransformed cells that grow uncontrollably. A tumor may be benign(benign tumor) or malignant (malignant tumor or cancer). Tumors includeleukemias, lymphomas, myelomas, plasmacytomas, and the like; and solidtumors. Examples of solid tumors that can be treated according to theinvention include sarcomas and carcinomas such as, but not limited to:fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, rhabdomyosarcoma, myosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, basal cell carcinoma, epidermoidcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medullablastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, ogliodendroglioma, meningioma, melanoma, neuroblastoma,neuroglioma, and retinoblastoma.

In some embodiments, the cancer tropism of the nanospheres describedherein can be used to target therapeutic agents to cancer tissue withoutrequiring specific targeting agents. In some embodiments, the cancertropism of the nanospheres described herein can be used to targetdiagnostic agents to cancer tissue without requiring cancer specifictargeting agents.

In some embodiments, nanosphere particles are delivered to particularorgans or tissues (e.g., lung) or cells or subcellular locations.However, in some embodiments, nanosphere particles described herein canbe administered systemically and their natural tropism results in theirselective accumulation in cancer tissue.

Accordingly, in some embodiments, a therapeutically effective dose of ananosphere is one that is sufficient to result in a therapeutic level ofthe agent being delivered to the target tissue of interest (e.g., thecancerous tissue). In some embodiments, the amount of agent that isdelivered can be evaluated using one or more detectable agents (e.g., adetectable therapeutic agent, for example labeled with a detectablemoiety, or a detectable diagnostic agent) to determine the efficiency ofdelivery to the target tissue. In some embodiments, the nanosphere isadministered to a subject in an amount effective to treat a disease orcondition.

In some embodiments, the term “therapeutically effective amount” or“amount effective” in the context of a NSP or a NSP composition foradministration to a subject refers to an amount of the NSP orcomposition that ameliorates or treats the disease or condition.

Amounts effective will depend, in some embodiments, on the subject beingtreated; the severity of a condition, disease or disorder; the subject'sparameters including age, physical condition, size and weight; theduration of the treatment; the nature of concurrent therapy (if any);the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. In some embodiments, maximumdose, that is, the highest safe dose may be used according to soundmedical judgment. It will be understood by those of ordinary skill inthe art, however, that a subject may insist upon a lower dose ortolerable dose for medical reasons, psychological reasons or forvirtually any other reasons.

In some embodiments, the term “treat,” “treating,” or “therapeutic,” donot necessarily mean total cure or abolition of the disease orcondition. In some embodiments, any alleviation of any undesired signsor symptoms of a disease or condition, to any extent, can be consideredtreatment or therapy. In some embodiments, treatment may include actsthat may worsen the patient's overall feeling of well-being orappearance.

In some embodiments, the nanospheres are administered intravenously,intramuscular, subdermally, orally, nasally or topically. Other modes ofadministration may be used.

In some embodiments, provided herein are methods for selectivelydelivering an agent to a tumor in a subject, the method comprisingadministering a tumor tropic nanosphere particle to a subject, whereinthe tumor tropic nanosphere particle is associated with an agent, and isfree of host cell and viral nucleic acid, and wherein the tumor tropicnanosphere particle comprises one or more viral capsid proteins withouta heterologous targeting agent. In some embodiments, the agent is atherapeutic agent. In some embodiments, the therapeutic agent is aninorganic molecule, an organic molecule, or a biologic active molecule.In some embodiments, the therapeutic agent includes a small molecule, aprotein, a peptide, an antibody, a toxin, a nucleic acid, aradioisotope, a metal, an inducer of DNA methylation, a modulator ofgene expression, an immune modulator, an enzyme inhibitor, a kinaseinhibitor, an apoptosis inducer, a metabolism inhibitor, or anycombination thereof. In some embodiments, the nucleic acid is an siRNAmolecule, an shRNA molecule, a microRNA, a long non coding RNA, a hybridDNA-RNA, a DNA molecule, an antisense molecule, a viral gene cassette,or any combination thereof. In some embodiments, the therapeutic agentis a radioisotope or a radiolabeled molecule. In some embodiments, theagent is a diagnostic agent. In some embodiments, the diagnostic agentis an imaging or contrast agent. In some embodiments, the diagnosticagent is labeled with a detectable label. In some embodiments, thedetectable label is a fluorescent or radioactive label.

In some embodiments, the agent is encapsulated with the nanosphereparticle. In some embodiments, the agent is mixed with the viral capsidproteins in the nanosphere particle. In some embodiments, the agent ischemically linked to an amino acid of one or more capsid proteins in thenanosphere particle.

In some embodiments, the nanosphere particle is assembled from one ormore viral capsid proteins or capsomeres that are isolated and purifiedfrom a host cell expression system. In some embodiments, two differentviral capsid protein types are expressed from different nucleic acidconstructs in the host cell expression system. In some embodiments, theone or more viral capsid proteins and capsomeres are reassembled invitro to form virus like particles. In some embodiments, the host cellexpression system is bacterial, yeast, insect, plant or mammalian hostcell expression system. In some embodiments, the host cell expressionsystem is E. coli. In some embodiments, the nanosphere particle isassembled from one or more viral capsid proteins isolated from a cellfree expression system.

In some embodiments, the nanosphere particle comprises one or morecapsid proteins from Herpes Simplex Virus, Polyomavirus, PapillomaVirus, Epstein Barr Virus, Rous Sarcoma Virus or Rotavirus. In someembodiments, the nanosphere particle comprises one or more PapillomaVirus capsid proteins.

In some embodiments, the nanosphere particle comprises one or morecapsid proteins having an amino acid sequence alteration that modifiesthe immunogenicity of the capsid protein relative to anaturally-occurring capsid protein in the subject. In some embodiments,the capsid protein is a PV capsid protein having mutations that reduceor modify an PV serotype-specific immunogenicity in the subject. In someembodiments, the PV capsid protein is an L1 and/or L2 capsid protein. Insome embodiments, the nanosphere particle comprises a PV L1 capsidprotein without any L2 capsid protein. In some embodiments, at least oneviral capsid protein is modified to prevent an immunogenicity responsein the host. In some embodiments, the modification is a PEGylation.

In some embodiments, the subject has cancer, the agent is an anti-canceragent, and the nanosphere particle is administered in an amountsufficient to deliver a therapeutically effective dose of the agent tothe cancer. In some embodiments, the cancer is a solid-tumor with orwithout metastases and with or without cancer stem cells. In someembodiments, the disease is a pre-malignant tumor. In some embodiments,the agent is a diagnostic agent and the subject is being screened forone or more indicia of cancer. In some embodiments, the cancer isselected from the group consisting of leukemias, lymphomas, myelomas,plasmacytomas, sarcomas, carcinomas, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,rhabdomyosarcoma, myosarcoma, colon carcinoma, pancreatic cancer, breastcancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basalcell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat glandcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medullablastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, ogliodendroglioma, meningioma,melanoma, neuroblastoma, neuroglioma, and retinoblastoma.

In some embodiments, the cancer is present at two or more separatelocations in the subject.

In some embodiments, provided herein are methods for preparing ananosphere particle for selectively delivering a therapeutic ordiagnostic agent to a cancer in a subject, the method comprising, obtainviral capsid proteins without any host or viral nucleic acid, andreassembling the capsid proteins in the presence of a therapeutic ordiagnostic agent. In some embodiments, the capsomere is obtained from ahost cell expression system and purified. In some embodiments, thecapsid proteins are from a tumor tropic virus. In some embodiments, thetumor tropic virus is PV. In some embodiments, the capsid proteincontains an amino acid sequence alteration or amino acid modificationthat changes the immunogenicity of the nanosphere particle in thesubject.

In some embodiments, PV L1 and L2 capsid proteins are used. In someembodiments, PV L1 capsid protein is used, but no L2 capsid protein isused.

In some embodiments, provided herein are methods of producing humanpapillomavirus (PV) nanosphere particles loaded with an agentcomprising: recombinantly expressing mutant PV L1 and wild-type PV L2capsid proteins, or mutant PV L1 without any PV L2 capsid proteins invitro in E. coli cells, wherein the mutant PV L1 capsid protein hasmutations that differs from the wt HPV; isolating the L1 and L2capsomeres, or L1 capsomeres; combining the capsid protein capsomereswith the agent and reassembly buffer containing salt and HEPES buffer,or salt and Histidine-HCl; and dialyzing the combination of protein andbuffer to produce HPV nanosphere particles loaded with the agent. Insome embodiments, the reassembly buffer contains 0.5M NaCl, 5 mM CaCl2,and 40 mM HEPES (pH 6.8), or (2) 0.5M NaCl, 5 mM CaCl2, and 40 mMHistidine-HCl (pH 5.2).

In some embodiments, provided herein are methods of delivering andevaluating a cancer therapy comprising: a) identifying a subject withcancer; b) labeling tumor tropic nanosphere particles; c) loading thetumor tropic nanosphere particles with a therapeutic agent; d)administering a detectable amount of the nanosphere particles to thesubject; and e) determining the presence or amount of the nanosphereparticles in the subject during and after a period of a treatment. Insome embodiments, the label is selected from the group consisting offluorescent, radioactive, and chemiluminescent. In some embodiments, thenanosphere particle comprises a variant PV-L1 protein having amutation(s) that makes it immunogenically different from the wt HPV-16serotype.

Provided herein are compositions for the treatment or diagnosis ofcancer cells, the composition comprising a therapeutic or diagnosticagent formulated with a nanosphere particle, wherein the nanosphereparticle comprises structural proteins from HSV, RSV, Polyoma, PV,Epstein Barr or Rotavirus.

Also provided herein are compositions for the treatment or diagnosis ofcancer cells, the composition comprising a therapeutic or diagnosticagent formulated with a nanosphere particle, wherein the nanosphereparticle comprises a mutated or modified PV L1 protein, wherein themutation or modification reduces or modifies the PV serotype specificimmunogenicity of the nanosphere particle. In some embodiments, themutation or modification reduces or modifies HPV-16 serotype specificimmunogenicity.

EXAMPLES Example 1

Production of Mutant L1* and L2 Capsid Proteins in E. coli Cell System(FIG. 2A)

Purification of VLPs by sucrose gradient centrifugation

Make a stock solution of 65% sucrose by dissolving 32.5 g of crystallinesucrose (Fisher cat. #57-50-1) to a final volume of 50 ml sample buffer.Sample buffer used for VLP purification is 0.5M NaCl (AmericanBioanalytical cat. # AB01915) in sterile 1×PBS (Boston BioProducts cat.# BM 220S).

Make different concentrations of sucrose solution as described in Table1 by mixing appropriate volumes of 65% sucrose stock solution (Step 1)in sample buffer.

TABLE 1 Final ml sucrose 65% ml % stock buffer 50 7.69 2.31 40 6.15 3.8530 4.62 5.38 20 3.08 6.92 10 1.54 8.46

Gently overlay decreasing concentrations of sucrose (highestconcentration at the bottom) in a Beckman Polyallomer centrifuge tube(Cat. #326819). The volumes of different sucrose concentrations in thetube are as follows:

65% 0.5 ml 50% 0.5 ml 40% 0.75 ml 30% 0.75 ml 20% 0.75 ml 10% 0.75 ml-1ml

Keep the gradient undisturbed at room temperature for 45 min. Gentlyload clarified lysate supernatant on top of the sucrose gradient withoutdisturbing the layers below.

Centrifuge the tubes at 45,000 rpm at 4° C. for 2 hrs in a SW55Ti rotor(Beckman Coulter, Inc.).

Gently remove the tubes from the rotor and collect 0.2 ml fractions frombottom of the centrifuge tube. Analyze fractions by SDS-PAGE and BCAassay for total protein.

Purification of NSPs Using Heparin HiTrap Column

After first centrifugation, if the homogenate is stillturbid—re-centrifuged at 15,000 g for 30 min.

Recover clarified homogenate from and store at −80° C. until use.

Add 0.01% Tween 80 to clarified homogenate.

Dialyze into PBS supplemented to 0.25 M NaCl, 2 mM DTT, 0.01% Tween 80,pH 7.4—overnight at 4° C. with three changes of buffer.

Equilibrate 1-mL HiTrap Heparin HP with 10 column volumes (CV) ofdialysis buffer.

Load entire volume of dialyzed homogenate onto Heparin column at −0.1mL/min.

After loading, chase sample with ˜2 CV of dialysis buffer.

Elute column with step gradient of increasing NaCl concentration—allsteps contain PBS plus 1 mM DTT, 0.01% Tween 80-2.5 CV of each step:0.4, 0.6, 0.8, 1.0 & 1.5 M NaCl.

Collect 1.0 mL fractions of flow-through from loading and 0.5-mLfractions during elution.

Determined absorbance of fractions at 260, 280 & 340 nm.

Analyze load flow-through and NaCl gradient elution fractions byreducing SDS-PAGE on Bio-Rad TGX Any kD gels—stained with CoomassieR-250.

Purification of NSPs by Size-Exclusion Chromatography

Preparation of an agarose gel filtration column.

De-gas the DPBS-BSA solution by exposure to vacuum.

Clamp the column to a ring stand. Put the bottom cap on and add 5 ml ofDPBS/0.5 M NaCl.

Remove the bottom cap to eject any bubbles. Recap and add more DPBS/0.5M NaCl. Fill to near the top of the column.

Float a frit on the surface. Gently tap the frit to dislodge any airbubbles. Tap frit down to the bottom of the column using a 1- or 5-mlpipet (or the serum separator).

Remove the bottom cap and drain out most of the fluid.

Suspend the agarose beads by gently swirling and inverting the bottle.Pour bead slurry into the column. Fill the column to the rim.

Remove the bottom cap. Partially exchange the beads intoroom-temperature DPBS-BSA by repeatedly allowing the column to drip tonear dryness then pouring on more DPBS-BSA.

Replace the bottom cap. Cover the top of the column with Parafilm.Suspend beads by repeated gentle inversion of the column. Return thecolumn to the clamp and allow blocking and settling overnight at roomtemperature.

Remove Parafilm. Float a frit on the fluid surface and gently tap downto within a few mm of the bed surface.

Remove the cap from the bottom of the column. Wash the column with atleast 10 column volumes of DPBS/0.5 M NaCl.

Optional: If capsids are being purified out of crude cell lysate add 1μl of Benzonase nuclease and incubate 10 to 30 min at 37° C. to digestany residual unencapsidated DNA.

Add 0.5 ml or less (i.e., less than 1/10 of the agarose bed volume) ofclarified lysate (or capsids in Optiprep) to the washed agarose gelfiltration column.

Apply 0.25 ml of DPBS/0.5 M NaCl to the top of the column. Collectcolumn eluate in a siliconized 1.5-ml tube. Repeat this for a total of12 0.25-ml fractions.

Screen fractions for encapsidated DNA and protein.

Regenerate columns for re-use by washing the column with 10 columnvolumes of DPBS/0.5 M NaCl, then exchanging into DPBS-BSA supplementedwith 0.05% (w/v) NaN3 or other preservative. Store the column at roomtemperature for several days.

After column purification samples were analyzed by Electron Microscopyshowing assembled particles (FIG. 3B).

Example 2 Production of Mutant L1* and L2 Capsid Proteins in MammalianCell System (FIGS. 4A & B)

Plasmids containing human-optimized codon sequences were used to produceHPV16/31L1 mutant (L1*) and a HPV16L2 capsid proteins using a mammaliancell culture system, as follows.

HPV L1/L2 nanosphere particles and L1 only nanosphere particles wereadded to disassembly buffer (0.5 M NaCl, 10 mM DTT (Dithiothreitol), 20mM EDTA (Ethylenediaminetetraacetic acid), 50 mM Tris-HCl (pH 8.0)) toreach a final concentration of 0.05 mg/ml. The solution was incubatedovernight at 37° C.

The HPV nanosphere particles were then concentrated to between 0.1-0.2mg/ml using 100 kD spin columns. DNA or siRNA was added at a ratio of 1μg of nucleic acid for every 5 μg of nanosphere particle. The resultantmixture was dialyzed using 100 kD cut-off dialysis tubing against one oftwo reassembly buffers: (1) 0.5M NaCl, 5 mM CaCl2, 40 mM HEPES (pH 6.8);or (2) 0.5M NaCl, 5 mM CaCl2, 40 mM Histidine-HCl (pH 5.2). Thereassembly buffer was changed three times overnight (˜12-16 hours)during the dialysis.

To remove free (unencapsidated) DNA, the reassembled nanosphereparticles were treated with Benzonase endonuclease (1 U/μg of input DNA)for 10 minutes at room temperature. The Benzonase endonuclease wasremoved by one of three ways: (1) chemical inactivation using 0.5 MNaCl+80 mM EDTA for 1 hour at room temperature; (2) column removal usinga 100 kD spin column to remove the endonuclease and replace the volumewith an appropriate reassembly buffer; or (3) purification over anOptiprep (Iodixanol) gradient, following methods described (Buck andThompson, Current Protocols in Cell Biology, December 2007,26.1.1-26.1.19).

Removal of free DNA was achieved in one of two ways: (1) column removalusing a 100 kD spin column to remove the free DNA and replace the volumewith appropriate reassembly buffer; or (2) purification over anIodixanol gradient, following methods described (Buck and Thompson(2007)).

Quantitation of encapsidated or nanosphere particle-associated nucleicacid was achieved by: (1) digesting the particles in buffer containing0.01 U Proteinase K, 0.25% SDS, and 25 mM EDTA to liberate the nucleicacid from the protein (Buck and Thompson (2007)); (2) running DNAsamples on a 1% agarose gel (TAE buffer+GelRed dye), or running siRNAsamples on a 3% agarose gel (TAE Buffer+GelRed dye) with a molecularweight (MW) marker and a standard of the same encapsidated materialranging from 1 μg to 1 ng; and (3) quantitating the liberated nucleicacid using Image J software (Buck and Thompson (2007)).

Loading Protocol of negatively charged drugs by passive diffusion insideparticles: Disassembly protocol for modifying open pores with nodisassembly, followed by drug and closing of the pores is the samedisassembly protocol described above except that the incubation periodis lessened to 2 hours at room temperature instead of overnight at 37°C.

Loading of drugs by chemical conjugation (iodine): Iodixanol iseliminated to enable appropriate binding of Iodine, followed bypurification methods. Particles can be purified using a Cesium Chloridegradient, a sucrose gradient, or agarose gel filtration in lieu ofIodixanol. Binding of iodine is done to the exposed histidines in thestructure of the protein (FIG. 4B).

Example 3

Biodistribution Study in Parental SKOV3 Orthotopic Tumor Model withSpontaneous Metastasis (FIGS. 5A &B)

An orthotopic murine model for ovarian cancer with spontaneousmetastases was used to compare the specificity and efficiency of thenanosphere particle of the present invention with a human papillomavirus (HPV) virus-like particle (VLP) (also referred to herein as PsVparticles) for measuring biodistribution. Three groups of severecombined immunodeficiency (SCID) female mice received implantation ofSKOV3 (ATCC) parental tumor cell line by unilateral ovarian graft. Theexperiment was designed to compare HPV pseudovirus (PsV) particles tothe nanosphere particles of the present invention. Subjects in group 3received a single intraperitoneal injection of nanosphere particles(0.65 ml) when tumor sizes reached medium to large by palpitation on day77 post-tumor implantation.

FIG. 5A shows the results of in vivo bioluminescent signals as 48 hoursafter dosing. FIG. 5B shows the results of ex vivo tissue bioluminescentimaging of the primary tumor and tumors metastasized to the lung, theliver, the spleen GI-LN, the femur and the brain at 48 hours postdosing.

Example 4 Biodistribution Study in SKOV-3 Mice Comparing Pseudovirionsto Nanosphere Particles (FIGS. 6A & B)

In comparing the total luminescence shown between the negative controlgroup, the PsV particles, and the nanosphere particles, the nanosphereparticles according to the present invention were shown to producebetter distinction which is evidence of a better treatment option. Themethod of producing forming nanosphere particles eliminates thepossibility of introducing host cell DNA. Also, the nanosphere particlesexhibit inherent tumor tropism. Thus, the nanosphere particle is acleaner, more efficient vector for delivery of therapeutic anddiagnostic agents, as compared to existing virion-derived particles.

Protocol (FIG. 4A)

For 12 mice receiving PsV particles, a 0.5 μg DNA equivalent isdelivered for the following constructs:

16modLuc (Luciferase expression, 9.9.11RK)−0.43 ng DNA/10 μl→0.5 μg=11.6μl/mouse (an amount sufficient for 16 mice should be made to account forloss: 185.6 μl PsV for 16 mice).

16modRwB (Red fluorescent protein (RFP) expression, 9.9.11RK)−0.41 ngDNA/10 μl→0.5 μg=12.2 μl/mouse (an amount sufficient for 16 mice shouldbe made to account for loss: 195.2 μl PsV for 16 mice).

A preparation is made by combining 185.5 μl of Luc with 195.2 μl of RwBHPV=380.7 μl. This is diluted with 1.6193 mls of sterile DPBS for aVT=2.0 mls. This is mixed well immediately prior to injection, deliver125 μl/mouse is delivered.

For 20 mice receiving nanosphere particles, a 1 μg DNA equivalent isdelivered.

An amount sufficient for 25 mice should be made to account for loss (for25 mice, the amount should be doubled just in case of loss, so an amountsufficient for 50 mice should be made). The current loading methodappears to yield an approximate 50% recovery of loaded DNA, therefore todeliver 1 μg, sufficient “doses” should be made to provide 2 μg/mouse.

A ratio of 5 μg protein: 1 μg DNA is used (for 50 mice, 500 μgprotein:100 μg DNA is used). The following steps are performed:

Day one—Disassembly using HV16mod L1/L2 VLPs (RK 09.16.11@1.1 mg/ml).

VLPs are disassembled in 50 mM Tris (pH 8.0)/0.5 M NaCl/20 mM EDTA/10 mMDTT—Protein CF=0.05 mg/ml in 20 ml 0/N @ 37° C.

Day two—DLS to ensure disassembly—notice some aggregates—60 secondssonicate.

Using a 100 kD spin column, disassembled particles are concentrated downto 0.2 mg/ml (5 mls). The result is split into two reactions—2.5 mlseach. The disassembled particles are combined with 100 μg of plasmid DNAand placed into a 5 ml float-a-lyzer (100 kD). For pCLucF plasmid stockat 0.9 mg/ml, a 111 μl volume is used for 100 μg. For pRwB plasmid stockat 1.3 mg/ml, a 77 μl volume is used for 100 μg. This is dialyzedagainst 40 mM HEPES (pH 6.8)/0.5 M NaCl/5 mM CaCl2—overnight at roomtemperature, with three buffer changes.

Day three—Samples are collected and analyzed for DNA and proteincontent.

16modLuc (10.18.11DD)—0.175 ng DNA/10 μl→1.0 μg=57 μl/mouse. Make enoughfor 25 mice to account for loss. 1.425 ml HPV Luc for 25 mice. 16modRwB(10.18.11DD)—0.197 ng DNA/10 μl→1.0 μg=50.8 μl/mouse. Make enough for 25mice to account for loss. 1.27 ml HPV RFP for 25 mice. Combine 1.425 mlof Luc with 1.27 ml of RFP HPV=2.695 ml. Diluted with 0.43 μl of sterileHEPES buffer for a VT=3.125 mls. For the best results, it is imperativeto mix well immediately prior to injection—deliver 125 μl/mouse.

Day four—Mice are injected.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present invention is notintended to be limited to the above Description, but rather is as setforth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the listed claims is introduced into another claim. For example,any claim that is dependent on another claim can be modified to includeone or more limitations found in any other claim that is dependent onthe same base claim.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., some embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is also noted that theterm “comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any some embodiment of thepresent invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anysome embodiment of the methods of the invention can be excluded from anyone or more claims, for any reason, whether or not related to theexistence of prior art.

Each of the foregoing patents, patent applications and references ishereby incorporated by reference, particularly for the teachingreferenced herein.

1-38. (canceled)
 39. A nanosphere particle comprising a papillomavirusL1 capsid protein encoded by the nucleic acid sequence of SEQ ID NO: 2.40. The nanosphere particle of claim 39, wherein the nanosphere particleis associated with an agent.
 41. The nanosphere particle of claim 40,wherein the agent is a therapeutic agent.
 42. The nanosphere particle ofclaim 41, wherein the therapeutic agent is an inorganic molecule, anorganic molecule or a biologic active molecule.
 43. The nanosphereparticle of claim 41, wherein the therapeutic agent comprises a smallmolecule, a protein, a peptide, an antibody, a toxin, a nucleic acid, aradioisotope, a radiolabeled molecule, a metal, an inducer of DNAmethylation, a modulator of gene expression, an immune modulator, anenzyme inhibitor, a kinase inhibitor, an apoptosis inducer, a metabolisminhibitor, or any combination thereof.
 44. The nanosphere particle ofclaim 41, wherein the therapeutic agent is an anticancer agent.
 45. Thenanosphere particle of claim 41, wherein the therapeutic agent is atoxic agent.
 46. The nanosphere particle of claim 40, wherein the agentis a detectable moiety.
 47. The nanosphere particle of claim 46, whereinthe detectable moiety is fluorescent.
 48. The nanosphere particle ofclaim 40, wherein the agent is attached to a surface of the nanosphereparticle.
 49. The nanosphere particle of claim 39, wherein thenanosphere particle is free of host cell nucleic acid and viral nucleicacid.
 50. The nanosphere particle of claim 39 further comprisingpapillomavirus L2 capsid protein.
 51. The nanosphere particle of claim39, wherein the modified papillomavirus L1 capsid protein has at leastone amino acid that is PEGylated.
 52. The nanosphere particle of claim39, wherein the nanosphere particle is assembled in vitro from aplurality of modified papillomavirus L1 capsid proteins encoded by thenucleic acid sequence of SEQ ID NO:
 2. 53. The nanosphere particle ofclaim 48, wherein the agent is an anticancer agent.
 54. The nanosphereparticle of claim 48, wherein the agent is a toxic agent.
 55. Thenanosphere particle of claim 53 further comprising papillomavirus L2capsid protein.
 56. The nanosphere particle of claim 54 furthercomprising papillomavirus L2 capsid protein.
 57. A papillomavirus L1capsid protein encoded by the nucleic acid sequence of SEQ ID NO:
 2. 58.A nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 2.